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Clinical Science (2012) 122, 397–407 (Printed in Great Britain) doi:10.1042/CS20110506 397

R E V I E W

Endothelial cells and magnesium: implications

in atherosclerosis

Jeanette A. M. MAIER
Dipartimento di Scienze Cliniche L. Sacco, Università di Milano, Via G.B. Grassi 74, Milano, Italy

A B S T R A C T

Clinical Science
There is no doubt that the functional and structural integrity of the endothelium is critical in
maintaining vascular homoeostasis and in preventing atherosclerosis. In the light of epidemiological
and experimental studies, magnesium deficiency is emerging as an inducer of endothelial
dysfunction. In particular, data on the effects of low extracellular magnesium on cultured
endothelial cells reinforce the idea that correcting magnesium homoeostasis might be a helpful
and inexpensive intervention to prevent and treat endothelial dysfunction and, consequently,
atherosclerosis.

INTRODUCTION endothelial phenotype by various noxious stimuli. As a

Despite considerable advances over the last decades,
cardiovascular diseases exact a very high toll as the leading
cause of death in Western societies. This is mainly the
result of the increasing prevalence of atherosclerosis,
related to the aging of the population, the increase in
diabetes and obesity, unhealthy diets, and the under-
recognition and under-control of different risk factors
for atherosclerosis, in particular hyperlipaemia and
hypertension [1].
Atherosclerosis is considered to be a chronic inflam-
matory disease resulting from the interaction between
modified lipoproteins, monocyte-derived macrophages,
and endothelial and smooth muscle cells [2]. Initially,
this inflammatory process results in the thickening of
the arterial wall and then in the formation of complex
lesions, the atherosclerotic plaques, through a process
that occurs slowly and silently over decades. The plaques
protrude in the arterial lumen and, when complicated,
cause myocardial infarction, stroke and gangrene [1,2].
Atherosclerosis is initiated upon the modulation of the
consequence, endothelial cells enable infiltration and re-
tention in the intima of LDLs (low-density lipoproteins),
the major cholesterol-carrying lipoprotein in plasma, and
contribute to their oxidation. In addition, endothelial cells
express adhesion molecules which recruit monocytes. In
the vessel wall, monocytes mature into macrophages and
take up modified LDLs to the point that their cytoplasm
is engorged with cholesterol, generating the so-called
foam cells [2], which are crucial to plaque progression in
association with smooth muscle cells migrating from the
media to the intima. The ongoing inflammatory response
sustains and amplifies the process and has a prominent
role in driving the complications of the plaque [2].
The treatment of hypercholesterolaemia and hyper-
tension 20–30 years ago was expected to eliminate
cardiovascular diseases by the end of the 20th century,
but the pathogenic picture has become increasingly
complex and such an optimistic prediction needs revision.
Thus an understanding of other factors contributing to
atherogenesis is necessary in the development of new
strategies for prevention and treatment.

Key words: atherosclerosis, endothelial cell, inflammation, magnesium, oxidative stress, thrombosis.
Abbreviations: apoE, apolipoprotein E; EPC, endothelial progenitor cell; GM-CSF, granulocyte/macrophage colony-stimulating
factor; HDL, high-density lipoprotein; hsp, heat-shock protein; IL, interleukin; LDL, low-density lipoprotein; MMP, matrix
metalloprotease; NF-κB, nuclear factor κB; IκB, inhibitor of NF-κB; ox-LDL, oxidized LDL; PAI-1, type 1 plasminogen activator
inhibitor; PDGF, platelet-derived growth factor; RANTES, regulated upon activation, normal T-cell expressed and secreted; ROS,
reactive oxygen species; TRPM, transient receptor potential melastatin; VCAM, vascular cell adhesion molecule; vW, von Willebrand.
Correspondence: Dr Jeanette A.M. Maier (email jeanette.maier@unimi.it).

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MAGNESIUM AND CARDIOVASCULAR
DISEASES
Environmental factors are fundamental in atherogenesis
and, accordingly, changes in lifestyle have yielded excel-
lent results. Intervention on dietary habits is particularly
encouraging. There is a general agreement about the
inverse correlation between regular consumption of fruit,
cereals and vegetables and the risk of atherosclerosis
[3]. Edible plant matter contains many beneficial
microconstituents, polyphenols, vitamins and minerals.
In particular, Mg (magnesium), which is predominantly
obtained by eating unprocessed grains and green leafy
vegetables, is an essential micronutrient implicated in
a large array of regulatory, metabolic and structural
activities [4]. The Western diet is relatively deficient
in Mg because of low Mg content in water and soil,
the processing of many food items, and the preference
for calorie-rich and micronutrient-poor diets [5]. This
explains why Mg deficiency is relatively common in
industrialized countries. Inadequate dietary intake of Mg,
however, is not the only cause of Mg deficiency. Mg
homoeostasis is very tightly controlled in the intestine
and in the renal tubules through a complex network of
transporters, some of which have recently been defined
at the molecular level [6]. Consequently, Mg deficiency
accompanies chronic gastrointestinal and renal diseases,
and also complicates diabetes mellitus and therapies with
some classes of diuretics, antibiotics or antineoplastic
drugs. In addition, it is common in the elderly and in
alcoholics [5].
Clinical studies investigating the links between dietary
or serum Mg, vascular dysfunction and atherosclerosis
have yielded conflicting results. The NHEFS (National
Health Epidemiologic Followup Study) found no
significant association between serum Mg and the
incidence of cardiovascular diseases [7], and similar
results were reached in the Framingham Heart Study
offspring cohort [8]. However, it should be noted that the
measurement of serum Mg, although commonly used in
clinical practice, does not fully reflect its homoeostasis [9],
Mg being primarily an intracellular cation. In addition,
serum Mg should be measured more than once, because
of variations in Mg levels depending upon diet, alcohol
consumption and physical exercise [10].
In disagreement with the studies described above, the
ARIC (Atherosclerosis Risk in Communities) cohort
showed an inverse relationship between serum Mg and
the development of carotid atherosclerosis [11], and
the Paris Prospective Study 2 reported a relationship
between low serum Mg and cardiovascular mortality in
middle-aged men [12]. In addition, in patients on dialysis,
a condition that accelerates atherosclerosis, Mg has a
protective role in the progression of the disease [13]
and, in renal transplant recipients, hypomagnesaemia is
an independent predictor of arterial stiffness [14].
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When a link between dietary Mg intake and incidence
of cardiovascular diseases was investigated, an inverse
association was reported [15,16]. These results are
in keeping with the finding that hypomagnesaemia
induces a pro-atherogenic lipid profile by decreasing
HDLs (high-density lipoproteins) and increasing total
serum cholesterol, LDLs and triacylglycerols (trigly-
cerides) [17]. Interestingly, experimental data have
demonstrated that Mg deficiency decreases lipoprotein
lipase activity, which is implicated in the production
of HDL and in the catabolism of triacylglycerol-
rich lipoproteins [18], activates HMG-CoA reductase
(3-hydroxy-3-methylglutaryl-CoA reductase), the rate-
limiting enzyme in cholesterol synthesis, and LCAT
(lecithin:cholesterol acyltransferase), which catalyses the
formation of cholesterol esters [19]. Moreover, Mg acts on
the lipid profile indirectly, as it modulates insulin action
[17]. In particular, Mg deficiency impairs the tyrosine
kinase activity of the insulin receptor, an event related to
the development of post-receptor insulin resistance [17],
which is a well-recognized risk factor for atherosclerosis.
To this purpose, it is interesting to recall the recent
finding that bittern, a natural MgCl2 solution from the
sea widely utilized in Japan to coagulate tofu, can be
used as a natural Mg supplement to improve post-prandial
hyperlipidaemia in healthy adults [20].
Relevant to atherogenesis is the association of Mg
deficiency and oxidative stress [21]. This condition
facilitates the oxidation of LDLs, thus potentiating
their pro-atherogenic potential, and also promotes
inflammation through the activation of the redox-
sensitive transcription factor NF-κB (nuclear factor
κB), which induces cytokines, growth factors, adhesion
molecules and enzymes involved in inflammatory
responses [22]. It is noteworthy that an inverse
association between Mg intake and the CRP (C-reactive
protein) level, a marker of systemic inflammation and
a known risk factor for cardiovascular diseases, was
reported in different human studies [23–25], and Mg
supplementation in overweight individuals led to changes
in gene expression and proteomic profile consistent with
favourable effects on inflammation [24]. On the basis
of human studies, Figure 1 summarizes the mechanisms
involved in linking hypomagnesaemia or inadequate Mg
intake to atherosclerosis.
The findings from the population studies described
above are emphasized by the demonstration that experi-
mentally induced Mg deficiency promotes atherosclerosis
in several animal models, mainly through the activation of
inflammation and hyperlipaemia [26,27]. Hyperlipaemia
and inflammation are considered ‘partners in crime’,
since atherosclerosis is an inflammatory disease which
is initiated by and progresses in the context of
hypercholesterolaemia [28]. Therefore there is sufficient
evidence to consider Mg deficiency a multi-faceted
contributing factor to atherogenesis.

Figure 1 How low Mg intake and/or hypomagnesaemia
contribute to atherogenesis: a summary from human studies
Human studies indicate that low Mg intake and/or hypomagnesaemia promote
inflammation, oxidative stress, insulin resistance and hyperlipaemia, all known risk
factors for atherosclerosis.
THE ENDOTHELIUM IN ATHEROSCLEROSIS
Endothelial cells cover the entire inner surface of the
blood vessels and are fundamental for the integrity
of the vascular wall and for maintaining circulatory
functions. Far from being a passive barrier between
blood and tissues, the endothelium actively and reactively
regulates vascular tone by balancing the synthesis of
vasoconstrictors and vasodilators, controls blood fluidity
by synthesizing factors that regulate platelet activity and
fibrinolysis/coagulation, participates to the inflammatory
process by producing cytokines and adhesion molecules
that control the ingress and egress of leucocytes and
inhibits medial smooth muscle cell growth [29]. Not
surprisingly, therefore, endothelial function has been
suggested to serve as a barometer for cardiovascular
health [30].
In physiological conditions, the endothelium con-
tinuously monitors blood-borne and locally generated
stimuli by adaptively altering its functional state and
by responding to changes in the environment. In
response to noxae, such as risk factors for atherosclerosis,
the endothelium undergoes functional and structural
alterations that jeopardize its protective role and generate
a pro-atherogenic phenotype [31]. Damage to the
endothelium upsets the balance between vasoconstrictors
and vasodilators and triggers various processes that
initiate, promote or exacerbate atherosclerosis, such
as increased endothelial permeability, reduced NO
bioavailability, leucocyte adhesion and synthesis of
cytokines (Figure 2). This complex and dynamic process
Endothelial cells and magnesium: implications in atherosclerosis 399
is referred to as endothelial dysfunction. A huge amount
of experimental evidence supports the paradigm of
endothelial dysfunction as the common link between
risk factors and atherosclerotic burden. Endothelial
dysfunction is an early event in the atherogenic process
and precedes angiographic and ultrasonic evidence of
damage to the arterial wall [32]. It also contributes to
the later stages of the disease and plays a role in acute
coronary syndromes [30]. In clinical practice, numerous
methods have been employed to assess endothelial
function. Since studies indicate that endothelial function
detected non-invasively in the brachial artery correlates
with endothelial coronary function, brachial artery
ultrasound, which indirectly examines vasodilation in
response to stimuli that release NO [33], has emerged as
a useful tool for preventive or interventional approaches.
By utilizing this technique, it has been demonstrated
that interventions proven to reduce cardiovascular risk,
such as smoking cessation, lipid-lowering therapy, ACE
(angiotensin-converting enzyme) inhibitors and physical
exercise, improve endothelial function and decrease
cardiovascular risk [32,33]. It is worth highlighting that
endothelial function is also significantly correlated to Mg
levels and that Mg supplementation results in a significant
improvement in endothelial function, associated with
an amelioration of exercise duration in patients
with coronary artery disease and in diabetic individuals
[34–36].
These results in humans are supported by evidence that
Mg deficiency has an impact on vascular structure and
function in different experimental models. A moderate
long-term Mg-deficient diet increases intima-media
thickness and stiffness in rats due to increased collagen
content and a reduction in the elastin/collagen ratio [37].
In addition, endothelial function is significantly impaired
in a model of inherited hypomagnesaemia in mice, as
indicated by the decrease in endothelium-dependent
vasodilation, which associates with low plasma nitrate
levels [38].
Even though studies on cultured cells can only focus
on some isolated aspects of the complex events implicated
in the development of atherosclerotic plaques, they have
produced an extraordinary body of knowledge. It is
now time to turn our attention to how endothelial cells,
actively involved from the very early to the late stages of
atherogenesis, react to culture in low Mg.
MAGNESIUM AND THE ENDOTHELIAL CELL
Mg is the most abundant intracellular bivalent cation,
mostly complexed with ATP and other molecules. It
is involved in a wide variety of biochemical reactions
either by bridging distinct molecules or by functioning
as an allosteric modulator through its interaction with
negatively charged moieties [4].
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Figure 2 How low Mg contributes to endothelial dysfunction: evidence from in vivo and in vitro studies
Analogously to other risk factors for atherosclerosis, Mg deficiency promotes endothelial dysfunction. Under physiological conditions, the endothelium is a selective
barrier to the egress of molecules from the blood. It also maintains the balance between vasodilators and vasoconstrictors and between thrombotic and antithrombotic
factors. In addition, healthy endothelial cells do not interact with circulating cells. Low Mg upsets this balance, thus leading to endothelial dysfunction. On the contrary,
Mg supplementation in humans and in experimental models, as well as high extracellular Mg on cultured endothelial cells, rescue physiological endothelial function.

Mg homoeostasis in cells is tightly regulated by precise Recently, the ‘non-imprinted in Prader–Willi/Angelman’

control mechanisms operating at the level of Mg influx
and efflux across the plasma membrane, and at the level
of intracellular Mg buffering and organelle localization
[6]. Mg extrusion occurs against the electrochemical
gradient and is mediated through an Na (sodium)-
dependent transporter, namely the Na/Mg exchanger
driven by the Na gradient, and the Na-independent Mg
exchanger [6]. At the present time, these two channels
have not been cloned and, consequently, information
about their operation and expression are lacking in all
tissues. Genetic and electrophysiological approaches have
led to the identification of several Mg entry systems
[6]. The first molecularly defined components of the
mammalian Mg transport machinery are two cation
channels of the TRPM (transient receptor potential
melastatin) channel family, TRPM6 and TRPM7. They
show the unique functional duality of being an ion
channel and a kinase, because they possess an active
threonine/serine kinase at their C-terminus, which
belongs to the atypical family of eukaryotic α-kinases.
TRPM7 has a ubiquitous expression pattern, also being
expressed in the endothelium, and seems to play a
prominent role in intracellular Mg homoeostasis, whereas
TRPM6 mainly regulates Mg transport in the kidney and
in the gut [6,39]. The presence of functional TRPM7, but
not of TRPM6, channels in human endothelial cells has
been demonstrated, and a role of TRPM7 in regulating
endothelial proliferation and NO synthesis has been
described [40].
In various cell types, other proteins have been shown
to regulate Mg influx, including claudin/paracellin-1, Mg-
transporter subtype 1, SLC41A1 and SLC41A2 (solute
carrier family 41 subtype 1 and 2 respectively) and
the ACDP2 (ancient conserved domain protein 2) [6].
1 and 2 genes were identified as plasma membrane Mg
transporters [6]. The expression of all of these molecules
in endothelial cells, however, has not been investigated
yet. In addition, Mg is transported in and out of various
organelles, mainly mitochondria and Golgi complex. The
transporters involved in these pathways have only begun
to be identified.
The majority of mammalian cells retain their basal
intracellular Mg content unchanged even in the presence
of very low extracellular Mg levels [4,6]. Endothelial cells
are no exception. Intracellular Mg is not significantly
modulated in endothelial cells chronically exposed to
low concentrations of the cation [41]. It is noteworthy,
however, that Mg deficiency may determine fluctuations
in the intracellular content of other ions, namely loss of
K (potassium) and accumulation of Ca (calcium) and Na
[41]. These alterations might, in part, mediate low Mg
effects on endothelial cells.
Low Mg and endothelial cells: how it all
begins
A relevant issue is to consider how low Mg levels
affect endothelial function. There is clear evidence
that Mg deficiency, similarly to common cardiovascular
risk factors, promotes oxidative stress in various cell
types including endothelial cells [42,43]. Free radicals,
among which superoxide anions, related ROS (reactive
oxygen species) and NO, behave as a double-edged
sword. At high concentrations, they are hazardous and
damage all major cellular constituents. At moderate
concentrations, free radicals play an important role as
regulatory mediators in signalling processes, in spite of
their ephemeral nature [44,45]. With particular emphasis

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on the endothelium, free radicals have a role in regulating
signalling cascades that control vasomotor activity under
basal conditions. Excessive free radicals, however, activate
pro-inflammatory and pro-thrombotic pathways that
generate endothelial dysfunction [46].
Culture of endothelial cells in low extracellular
Mg rapidly and transiently induces ROS production
and reduces intracellular glutathione, a thiol-containing
tripeptide which plays an important protective role
against cellular oxidative injury by detoxifying free
radicals and lipid peroxides [42,43,47]. As a consequence
endothelial cells are more susceptible to oxidative injury
[43,48]. In parallel, early oxidative DNA damage which
resolves within a few hours has also been observed
[48]. The primary biochemical source of ROS in the
vasculature appears to be the membrane-associated
NADPH oxidase enzyme complex, which catalyses
the reduction of molecular oxygen using NADPH as
an electron donor, generating superoxide [49]. Various
cytokines and hormones important in the pathogenesis
of vascular diseases up-regulate NADPH oxidase activity
and superoxide production. Interestingly, culture in low
Mg also induces NADPH oxidase activity in endothelial
cells (J.A.M. Maier, unpublished work).
Endothelial cells respond to low-Mg-induced oxidat-
ive stress by up-regulating stress proteins, thus activating
the typical adaptive response to environmental stresses
of different kinds (J.A.M. Maier, unpublished work). It is
noteworthy that diverse risk factors for atherosclerosis,
among which hypertension, ox-LDL (oxidized LDL) and
mechanical stress, evoke stress protein overexpression
in endothelial cells [50]. In particular, in endothelial
cells cultured in low Mg, hsp (heat-shock protein)
70 is rapidly up-regulated (J.A.M. Maier, unpublished
work). Apart from functioning as a chaperone, hsp70
increases endothelial survival and protects these cells
from apoptosis by oxidants [51]. Interestingly, in apoE
(apolipoprotein E)-knockout mice, which spontaneously
develop atherosclerosis, hsp70 is expressed at sites prone
to lesion formation before macrophage infiltration has
occurred [52], and in human atherosclerotic plaques
hsp70 is elevated [50]. In recent years, it became evident
that hsps have pro-inflammatory activities and, therefore,
contribute to atherogenesis [50].
Stress proteins work in tandem with the ubiquitin–
proteasome pathway for protein quality control to
prevent the accumulation of damaged proteins [52].
Accordingly, an induction of proteasome activity has
been observed in endothelial cells in low Mg [53].
Taken together, these events contribute to reach a new
metabolic homoeostasis, so that the cells remain viable
and metabolically active, although they show clear
characteristics of dysfunctional cells. The induction of
proteasome activity might also be involved in accelerating
the activation of NF-κB in endothelial cells cultured in
low Mg (see below).
Endothelial cells and magnesium: implications in atherosclerosis 401
From oxidative stress to endothelial
dysfunction: the increase in permeability
and the activation of NF-κB
In endothelial cells, together with the initiation of the
stress response, low-Mg-induced oxidants trigger some
early events which are crucial in atherogenesis, i.e.
the increase in permeability and the activation of the
transcription factor NF-κB.
Under normal conditions, the endothelium, with its
intercellular tight junctional complexes, functions as a se-
lectively permeable membrane. The excessive generation
of free radicals by endothelial cells or other cell types has
been implicated in the increased permeability of vascular
endothelium to molecules which would not otherwise
cross the barrier. Accordingly, antioxidant strategies im-
prove endothelial barrier dysfunction [54]. Low extracel-
lular Mg increases endothelial permeability [55]. Specific-
ally, Mg deficiency enhances LDL transport across the en-
dothelial monolayer [56]. In the first hours this is mainly
due to the stimulation of energy-dependent transport,
whereas later energy-independent transport also plays a
role because of the formation of intercellular gaps. Once
LDLs reach the subendothelial space, they are trapped by
matrix proteoglycans and become an easy target for ox-
idant species generated by low-Mg-stressed endothelial
cells [57]. It is well known that ox-LDLs are more
atherogenic than native LDLs and promote endothelial
dysfunction [28,58]. This means that ox-LDL and Mg
deficiency might co-operate to alter endothelial function.
Recently, the activation of NF-κB by low extracellular
Mg has been demonstrated in endothelial cells [59]. NF-
κB is known to play an important role in guarding the
delicate balance of the atherosclerotic process as a direct
regulator of pro-inflammatory genes and as a regulator of
cell survival and proliferation. The potential importance
of NF-κB in endothelial cells is further underscored by
experiments showing that the endothelium of regions
prone to developing atherosclerosis show higher levels
of different components of the NF-κB system compared
with other less risky regions [60].
In resting cells, NF-κB is inactive and retained in the
cytosol because of its association with the inhibitory IκB
(inhibitor of NF-κB) proteins. Upon cytokine signalling,
innate or adaptive immune responses, or oxidative stress,
NF-κB activation is initiated. The phosphorylation of
IκBs is an essential step in the activation of the classical
NF-κB activation cascade. Phosphorylated IκBs are
polyubiquitinated and degraded by the proteasome.
Released p50 and p65 NF-κB subunits translocate to
the nucleus and bind to NF-κB motifs in target genes,
regulating their transcription. Many of NF-κB-regulated
genes have been implicated directly or indirectly in
atherosclerosis [58,60].
Mg-deficiency-induced oxyradicals activate NF-κB in
endothelial cells through the canonical pathway [59].
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These findings are corroborated by evidence that Mg
supplementation markedly attenuates NF-κB nuclear
translocation and protects IκB from degradation in LPS
(lipopolysaccharide)-treated endothelial cells [61]. As a
result of the activation of NF-κB in the endothelium,
low Mg induces the expression of several cytokines,
chemokines, growth factors and adhesion molecules,
which are critical in atherogenesis.
IL (interleukin)-1α: a fundamental
mediator of low-Mg effects in endothelial
cells
Central in mediating some of low-Mg effects on
endothelial cells is IL-1α, a target of NF-κB and
an inducer of NF-κB itself. IL-1α is markedly up-
regulated early after exposure to low-Mg-containing
medium [59]. A founding member of the IL-1 family
now comprising 11 members, IL-1α controls pro-
inflammatory responses to cell injuries by various stimuli
[62]. Animal experiments have clearly demonstrated
that IL-1s are major pro-atherosclerotic factors and,
accordingly, clinical studies have been initiated with anti-
IL-1-targeting antibodies [63].
In addition to binding its surface receptor, IL-1α
functions intracellularly, being translocated to the nucleus
and influencing transcription [62]. In particular, in
endothelial cells endogenous IL-1α must be transported
to the nucleus to inhibit cell proliferation, promote
cellular senescence and modulate mRNA expression
of hundreds of genes, including its own expression,
thus generating a positive-feedback loop that amplifies
the IL-1 response [64]. It is through the induction of
IL-1α that Mg deficiency retards endothelial growth
by delaying the transit through the G1 - and S-phases
of the cell cycle [59,65]. Although endothelial cells
are normally quiescent, impaired proliferative capability
might become a problem in the case of vascular injury,
when it is important to heal the damage to maintain
the integrity of the vascular wall. Indeed, endothelial
turnover is accelerated by cardiovascular risk factors,
including certain lifestyle factors (i.e. lack of exercise,
Western diet, pollution and smoking), some diseases (i.e.
diabetes and hypertension) and mechanical stress (i.e.
catheterization and shear stress) [31]. When this occurs,
the damaged cells are removed by the bloodstream and
replaced by regenerated endothelium formed mainly by
neighbouring cells freed from contact inhibition.
As IL-1α is responsible for endothelial senescence [66],
it is relevant that culture in low Mg promotes the acquisi-
tion of some features that are typically associated with
endothelial senescence [53]. Interestingly, endothelial
cells with senescence-associated phenotypes are present
in human atherosclerotic lesions and also contribute to
atherogenesis because of their altered gene expression
[67]. The induction of cell senescence by low Mg,
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however, is not restricted to endothelial cells [68]. Indeed,
inadequate Mg availability accelerates telomere attrition
and, consequently, in vitro aging of fibroblasts [69].
IL-1α also induces various chemokines and adhesion
molecules in vascular endothelial cells through the
activation of NF-κB, thus favouring the recruitment,
adherence and diapedesis of monocytes. In particular,
low Mg stimulates the secretion of IL-8 and RANTES
(regulated upon activation, normal T-cell expressed
and secreted), chemokines which are overexpressed in
human atherosclerotic lesions [70]. IL-8 is critical for
the chemotaxis and adhesion of monocytes to the
endothelial cells, pivotal events in initiating atherogenesis,
and also stimulates VSMC (vascular smooth muscle
cell) proliferation and migration, critical steps for the
progression of the plaque [70]. Similarly, RANTES
directs leucocyte attraction and is also involved in
neointima formation in atherosclerosis-prone mice [70].
Through the induction of IL-1α, low Mg also stimulates
monocyte/endothelial interaction by overexpressing
VCAM (vascular cell adhesion molecule)-1 on the
endothelial surface [53,65]. VCAM, up-regulated in
the arterial intima of human atherosclerotic plaques,
functions as both a scaffold for leucocyte migration and
a trigger of endothelial signalling through NADPH-
oxidase-generated oxidant species [71], thus exacerbating
the direct effects of low Mg on NADPH oxidase
activity. Free radicals induce signals for the opening
of intercellular passageways through which monocytes
migrate [44,45]. In addition, the secretion of GM-CSF
(granulocyte/macrophage colony-stimulating factor) is
markedly increased in Mg-deficient endothelial cells
[65]. GM-CSF regulates the differentiation of cells of
macrophage lineage, an event which takes place once
monocytes have reached the intima.
All these findings point to Mg deficiency as a
potential contributor to the accumulation of monocytes-
macrophages in the arterial wall during the early stages
of atherogenesis.
An inadequate availability of Mg also stimulates the
secretion of PDGF (platelet-derived growth factor)-BB
by endothelial cells [59,72]. To this purpose, it is worth
noting that PDGF-BB is a direct target of NF-κB, an
effect that can be potentiated by the presence of IL-
1α, which is known to stimulate PDGF production
[73]. However, it is also possible that PDGF induction
results from monocyte–endothelial interactions and is
mediated by adhesion molecules [74]. A huge amount of
experimental evidence is available about the contribution
of PDGF to atherogenesis [58]. First, PDGF expression is
increased in human atherosclerotic arteries [75]. Secondly,
PDGF, which is chemotactic and mitogenic for smooth
muscle cells, co-operates with other cytokines and
growth factors in driving the accumulation of these
cells in the intima, where they change phenotype, lose
myofilaments, enrich their content of Golgi and rough
 Endothelial cells and magnesium: implications in atherosclerosis 403

Figure 3 How low extracellular Mg affects cultured human endothelial cells: implications in the early phases of
atherogenesis
In vitro studies on endothelial cells have demonstrated that low Mg promotes the acquisition of a pro-inflammatory pro-atherogenic phenotype in endothelial cells.
Culture in low Mg leads to the production of free radicals which: (i) induce the expression of hsps involved in protecting the cells from death; (ii) increase endothelial
permeability, an event which facilitates LDL accumulation in the subendothelial space; and (iii) activate NF-κB, thus up-regulating adhesion molecules, cytokines,
chemokines and growth factors. In addition, the increased activity of the proteasome might contribute to the activation of NF-κB. The up-regulation of IL-1α has a
role in inducing endothelial senescence.

endoplasmic reticulum and synthesize proteins of the
extracellular matrix [58]. These events ultimately lead to
the formation of the fibrous plaque [58]. On the basis of in
vitro studies, Figure 3 outlines the principal mechanisms
through which low extracellular Mg impairs endothelial
behaviour.
Towards the end: low Mg as an inducer of
proteases and pro-thrombotic factors
Maintenance of the fibrous cap reflects the balance
between matrix production and degradation. An
active role of the endothelium in the remodelling
of extracellular matrix and, therefore, its potential
contribute to plaque instability have been described
[58]. In atherosclerotic plaques, the expression of MMPs
(matrix metalloproteases), which degrade collagen fibrils
leading to loss of fibrous cap integrity, is increased
[76]. In particular, a marked up-regulation of MMP-9
in unstable carotid plaques has been demonstrated and
genetic variations within the MMP-2 promoter region
have been associated with cap thickness, with consequent
influences on plaque vulnerability [76,77]. The expression
of these proteases is normally tightly regulated at
the transcriptional level. A second level of control
is represented by the requirement of the extracellular
activation of the latent pro-enzyme. A third level of
control on MMP activity involves the irreversible binding
of these proteases to their specific tissue inhibitors [TIMP
(tissue inhibitor of metalloproteinases)].
In endothelial cells cultured in Mg-deficient medium,
a marked increase in the total amounts and activity of
MMP-2 and MMP-9 has been reported [59]. Accordingly,
MMP-2 and MMP-9 determine the structural alterations
of the vascular wall in Mg-deficient rats [78].
An NF-κB-binding site is present in the promoter of
the MMP-9 gene [79]. It is therefore possible that low Mg
availability might directly increase MMP-9 expression
via NF-κB in the beginning and sustain it through IL-
1α later. The mechanisms underlying MMP-2 induction
by low Mg are still a matter of investigation. Regarding
the activation of the pro-enzymes, free radicals might
be involved. Indeed, oxyradicals are known to react
with thiol groups, including those preserving MMP
latency, and therefore activate MMPs [80]. In particular,
in cultured smooth muscle cells exposed to mechanical
stress, free radicals derived from NADPH oxidase
increase the activity of MMP-2 [81] and MMP-9 [82].
It is noteworthy that, in Mg-deficient endothelial cells,
MMP-2 and MMP-9 activity overrides the inhibitory
effect of TIMP-2 which is also induced [59], probably
as an attempt to counterbalance the detrimental effects of
the proteases.
Endothelial cells are pivotal in maintaining the balance
between anti- and pro-thrombotic factors [29]. Under
normal circumstances, endothelial cells actively prevent

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404 J. A. M. Maier
Figure 4 How low extracellular Mg affects cultured human endothelial cells: implications in the complications of the
atherosclerotic plaque
Through the activation of NF-κB and the increase in IL-1α, low extracellular Mg up-regulates PAI-1 and MMPs. Free radicals produced in excess might be involved in
the activation of proMMP-2 and proMMP-9 to the active enzymes. MMPs cause plaque instability, and elevated levels of PAI-1 are pro-thrombotic.
thrombosis by synthesizing molecules that block platelet
adhesion and aggregation, inhibit coagulation and lyse the
clots [29]. Healthy endothelium produces prostacyclin
and NO, which are co-released and form a particular
partnership to impede platelet adhesion [83]. Culture in
low Mg increases the endothelial synthesis of prostacyclin
[84], probably as the result of the induction of COX-
2 (cyclo-oxygenase-2) by free radicals and IL-1α. It
is interesting to note that this enzyme is expressed
and contributes to the increase in prostacyclins in
patients with atherosclerosis [85]. Low Mg also seems
to induce NO both in a model of Mg deficiency in
rodents [86] and in cultured endothelial cells [87], and
this might be explained within the framework of the
inflammatory response activated by low Mg. However,
the superoxide radical produced by NADPH oxidase
under Mg-deficient conditions reacts very rapidly with
NO, thus reducing NO bioactivity and generating
peroxynitrite, another reactive species with damaging
effects on macromolecules.
Low Mg also affects endothelial fibrinolytic activity.
Indeed, PAI-1 (type 1 plasminogen activator inhibitor),
which inhibits tissue plasminogen activator thereby
preventing fibrinolysis, is up-regulated in endothelial
cells in low Mg [65] and in the aortas of hypomagnesaemic
mice [38]. It is noteworthy that PAI-1 is induced
in inflammation and is overexpressed in senescent
endothelial cells [88], two features strictly associated
with Mg deficiency. High plasma levels of PAI-1 are
important in the pathogenesis of thrombotic disease
and an up-regulation of PAI-1 has been demonstrated
in human atherosclerotic lesions [89]. Accordingly,
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deletion of the PAI-1 gene completely reverses the
prothrombotic tendency of apoE-knockout mice despite
the persistence of excessive hypercholesterolaemia [90].
Figure 4 summarizes these results and links them to
their potential role in the complications of atherosclerotic
plaques.
In conclusion, Mg deficiency plays a role also in events
involved in the late phases of atherosclerosis by having
an impact on the endothelial synthesis of proteases and
pro-thrombotic factors.
Elevated extracellular Mg and endothelial
cells
All of the data available on cultured endothelial cells
highlight that low extracellular Mg markedly alters
endothelial function. The obvious question is whether
endothelial dysfunction is reversible and the answer
is yes. After culture in low Mg, the re-addition of
the cation to the medium to reach the physiological
concentration rescues the normal proliferation rate and
the physiological activities of endothelial cells, thus
indicating that no permanent modifications occur with
Mg deprivation [65].
To support findings showing that Mg supplementation
is beneficial in patients with coronary artery disease and
diabetes [34–36], evidence has been provided that culture
of endothelial cells in high Mg increases NO production
[91], prevents NF-κB activation and, therefore, the
induction of cytokines and chemokines [61], decreases
MMP-9 activity [92], and up-regulates proteolysis
of ultra-large vW (von Willebrand) factor [93], thus

inhibiting platelet adhesion (Figure 2). Accordingly, in
rats, the infusion of Mg blocks the formation of thrombi
after vascular injury by reducing platelet aggregation and
prolonging blood clotting time [94].
CONCLUSIONS AND FUTURE PERSPECTIVES
Studies on endothelial cells teach us that low Mg
promotes the acquisition of a pro-atherosclerotic and
pro-thrombotic phenotype by having an impact on events
involved in the initiation, progression and complications
of the plaque. In spite of the fact that some clinical
studies found no correlation between serum Mg and
cardiovascular disease [7,8], in vitro and in vivo studies
have clearly demonstrated that Mg deficiency not only
affects endothelial cells, but also smooth muscle cells
[95], thus highlighting the notion that an inadequate
availability of the cation targets two principal players in
atherogenesis.
New fascinating aspects of endothelial function
are emerging from research on EPCs (endothelial
progenitor cells) [96]. Recent insights indicate that
the injured endothelium can be regenerated by
circulating bone-marrow-derived EPCs, which accelerate
re-endothelialization and limit atherosclerotic lesion
formation. It is also clear that risk factors for atherosclero-
sis, among which age, hypertension, smoking, hypercho-
lesterolaemia and diabetes, reduce the number and func-
tional activity of these circulating EPCs, thus limiting the
regenerative capacity and contributing to atherogenesis
and atherosclerotic disease progression. At the moment
it is not known whether and how Mg deficiency affects
the number and the functions of EPCs. It is likely that
broadening our knowledge in this novel field, together
with deepening our understanding on the effects of Mg
and its transporters on the cells implicated in atherogen-
esis, will rejuvenate research in the field and, hopefully,
open new avenues to prevent and treat vascular diseases.
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C The Authors Journal compilation ⃝
C 2012 Biochemical Society