ARTICLE
Auteur(s) : Federica I Wolf1, Valentina
Trapani1, Matteo Simonacci1, Silvia
Ferré2, Jeanette AM
Maier2
1Istituto di Patologia Generale, e Centro di Ricerche
Oncologiche Giovanni XXIII, Facoltà di Medicina, Università
Cattolica del Sacro Cuore, Roma
2Dipartimento di Scienze Precliniche LITA Vialba,
Università di Milano, Milano, Italy
Endothelial cells cover the entire inner surface of the blood
vessels and play a crucial role in maintaining the functional
integrity of the vascular wall. Beyond their role in regulating
permeability, they are involved in the maintenance of a non
thrombogenic blood-tissue interface, in the modulation of blood
flow and vascular resistance, in the regulation of immune and
inflammatory reactions [1]. A huge amount of experimental evidence
supports the paradigm of endothelial dysfunction as the common link
between risk factors and atherosclerotic burden [2]. Indeed,
endothelial dysfunction actively participates in the process of
lesion formation by promoting early and late mechanisms leading to
atherosclerosis. These include the upregulation of adhesion
molecules with consequent leukocyte adherence, increased chemokine
secretion, increased cell permeability to lipids, enhanced LDL
oxidation, cytokine elaboration, vascular smooth muscle cell
proliferation and migration, and platelet activation [3].
Evidence has accumulated to suggest low magnesium (Mg) as
pro-atherogenic in vivo [4]. In vitro, extracellular Mg
concentrations play a critical role in modulating endothelial
activities, since Mg levels influence the synthesis of nitric oxide
[5], the uptake and metabolism of low-density lipoproteins [6], the
proliferation and gene expression of endothelial cells [7]. In
human endothelial cells we have also demonstrated that low Mg
promotes the acquisition of senescent features that might
contribute to atherogenesis [8].
Interestingly, low Mg has been associated with oxidative stress,
a crucial event in aging, atherosclerosis and other vascular
diseases [9, 10]. It is well known that reactive oxygen species
(ROS) are key mediators of signalling pathways that underlie
vascular inflammation in atherogenesis, starting from the
initiation of fatty streak development, through lesion progression,
to ultimate plaque rupture [9]. Moreover, ROS are responsible for
the age-related damage in the vascular endothelium [10]. In a
normal situation, a balanced-equilibrium exists among oxidants,
antioxidants and biomolecules. Excess of generation of free
radicals may overwhelm natural cellular antioxidant defences
leading to oxidation and further contributing to cellular
functional impairment.
Following this rationale and the abundant data of the literature
describing the influence of Mg on oxidative events, we undertook
the present work aimed at investigating whether oxidative stress
occurs in human endothelial cells acutely exposed to low Mg.
Materials and methods
Cell culture
Human umbilical vein endothelial cells (HUVEC) were cultured in
M199 containing 10% fetal calf serum, Endothelial Cell Growth
Supplement (ECGS) (150 μg/mL) and heparin (5 U/mL) on 2% gelatin
coated dishes [7]. The cells were routinely evaluated for the
expression of endothelial markers, i.e. endothelial nitric oxide
synthase, VE-cadherin and CD34, and utilized for 5-6 passages. All
culture reagents were from Gibco. A Mg free medium was purchased by
Invitrogen (Milano, Italy) and utilized to vary the concentrations
of magnesium by the addition of MgSO4. No significant
difference was observed when we used MgSO4 or
MgCl2 to add magnesium to the culture media (not shown).
In all the experiments the cells were seeded in growth medium;
after 24 h, the medium was changed to modulate extracellular
Mg concentrations from physiologic (1.0 mM) to low levels (0.1 mM).
In some experiments HUVEC were simultaneously treated with 20 ng/mL
of interleukin (IL) 1 and 6 (Preprotech, Rocky Hill, NJ, USA) for 1
or 4 h.
Intracellular ROS measurements
ROS formation was evaluated by intracellular dichlorofluorescein
(DCF) fluorescence. Confluent cells were exposed to low Mg for
various times, rinsed with PBS and loaded with 5 μM
H2DCF-DA (Molecular Probes, Leiden, The Netherlands) at
37°C in the dark for 20 minutes. 100 μM H2O2
were added in the last ten minutes. The blank was made by
incubating cells without DCF. Cells were trypsinised, resuspended
in PBS at 1 x 106/mL and analysed on a Coulter
EPICS 753 flow cytometer for DCF fluorescence (excitation 488 nm,
emission 530 nm).
Immunocytochemical analysis
Detection of 8-hydroxy-deoxyguanine (8-OHdG) coupled with
diaminobenzidine (DAB) (Vector, Burlingham, CA, USA) was carried
out as described [11]. HUVEC were grown on coverslips and directly
processed on this support. Semi-quantitative evaluation of the
nuclear staining was carried out by an optical microscope (ECLIPSE
E600, Nikon, Tokyo, Japan, at 400 x) connected to an Image-Pro plus
Version 4.1 (Media Cybernetics, Silver Spring, MD, USA). Nuclear
staining was evaluated in approximately 100 cells of randomly
chosen images by operators who were blind to the status of cell
treatment. Data are expressed as fold-increase compared to the
staining of control cells, namely a sample of cells before the
incubation in normal or Mg-depleted conditions.
Statistical analysis
Data are expressed as the mean ± SD of triplicates from at least
three separate experiments. For assessing significance, unpaired
Student’s t test was calculated and difference considered when p
< 0.05.
Results
Intracellular ROS in Mg deprived HUVEC
We have previously shown that low Mg impairs HUVEC function [7] and
promotes the acquisition of some features typical of senescence
[8], thus favouring atherogenesis [9]. Since oxidative stress has a
role in aging and in endothelial dysfunction [9, 10], we measured
intracellular ROS by DCF fluorescence in cells exposed to low Mg
(0.1 mM) vs controls (1.0 mM Mg) with or without treatment with
H2O2. Figure 1A shows that Mg
deprivation did not affect the basal levels of DCF-detectable ROS
in the first 6 hours, whereas at later times the levels of ROS
are slightly lower in Mg deprived cells compared to controls. As
expected, the addition of 100 μM H2O2
markedly increased DCF-detectable ROS from about 80-100 to 200-250
fluorescence units (F.U.) (figure 1B). Interestingly,
early after Mg deprivation (2-6 hours), the addition of
H2O2 seemed to unmask an increase of DCF
fluorescence compared to control (from about 200 to 250 F.U.). At
later time intervals, however, the DCF levels of Mg deprived cells
decreased below the controls.
Oxidative DNA damage in Mg deprived HUVEC
We also evaluated oxidative DNA damage by measuring 8-OHdG, the
most abundant guanine oxidation product. Figure 2A shows the net
increase of 8-OHdG levels after 2 h culture in 0.1 mM Mg both
under basal conditions and after exposure to
H2O2. After 24h of Mg deprivation, however,
the levels of 8-OHdG are comparable in cells in low Mg and
controls. Surprisingly, at this time point, the exposure to
H2O2 reverts the previous result as the level
of 8-OHdG is lower in Mg deprived cells than in the controls.
Figure 2B
correlates DCF-detectable ROS with 8-OHdG and shows that, after
exposure to H2O2, the levels of 8-OHdG are
always directly proportional to the amounts of the corresponding
DCF-detectable ROS.
Intracellular ROS in Mg-deprived HUVEC exposed to inflammatory
cytokines
Since in vivo Mg deficiency associates with the onset of an
inflammatory response leading to increased circulating levels of
cytokines [12], we studied whether low Mg affects endogenously
produced ROS in HUVEC treated with a mixture of the inflammatory
cytokines IL-1 and IL-6. 4 h exposure to IL-1 and IL-6
significantly increased the levels of DCF-detectable ROS (+20%) in
cells cultured in physiologic concentrations of Mg, but not in
cells cultured in 0.1 mM Mg (figure 3).
Discussion
The aim of this work is to understand whether oxidative stress is
involved in modulating Mg dependent HUVEC dysfunction in vitro.
We exposed HUVEC to low Mg for different times from 2 to
48 h and and observed no alterations of DCF-detectable
intracellular ROS under basal conditions. Interestingly, early
after exposure to 0.1 mM Mg, HUVEC were more sensitive to the
oxidant action of H2O2 than the cells
cultured in 1.0 mM Mg. Indeed, in the presence of
H2O2, we observed an increase of
DCF-detectable ROS in Mg-deprived cells compared to controls.
Similar results were obtained in other experimental models such as
myocardium and hepatocytes [13, 14]. This increase of ROS is
transient as it is followed by a stable decrease of
DCF-fluorescence below the levels measured in the controls. It is
possible that the addition of H2O2 unmasks a
border-line situation characterized by mild oxidative stress
induced by low Mg, which, however, does not overwhelm the
anti-oxidant capabilities of the cells. Indeed, we cannot exclude
that a modest increase of ROS after 2-6 h of Mg deficiency
might escape DCF detection due to the time and the duration of this
phenomenon. To explain the reduced amounts of DCF-detectable ROS
after longer exposure to 0.1 mM Mg, we hypothesize that the cells
might activate an adaptive response to counterbalance the excess of
ROS, as we recently demonstrated in murine mammary epithelial cells
which upregulate glutathione S-transferase activity (Wolf et al.,
unpublished).
We also evaluated oxidative DNA damage and observed higher
8-OHdG levels early (2 h) after Mg deprivation in respect to
the controls, both in basal conditions and after treatment with
H2O2. The increase of oxidative DNA damage
can be ascribed to an ongoing oxidative stress or to a transient
decrease of DNA repair systems. Indeed, low Mg may affect the
efficiency of DNA repair systems [15]. At later times, however, DNA
damage seems to correlate with the levels of ROS rather than with
Mg availability. We conclude that a rapid and transient oxidative
stress induced by low Mg might activate or potentiate antioxidant
defences which ultimately counteract increased ROS production and
consequent oxidative DNA damage.
In contrast with our results, an increased oxidative stress
associated to Mg deprivation has been described in endothelial
cells and other cell types such as hepatocytes, myocardiocytes,
skeletal muscle and red blood cells [16-22]. Most of these results,
however, have been obtained in conditions of a Mg deficiency
associated with a severe pro-oxidant stimulus, such as
dihydroxyfumarate/ADP-Fe3+ triggering lipid peroxidation
in endothelial cells and erythrocytes [19, 20] or serum-free
incubations in endothelial cells and hepatocytes [16, 21]. On these
bases, it is possible to conclude that a severe oxidative stress
can be exacerbated by Mg deprivation. Other works, however, report
conflicting results on the induction of oxidative stress and on the
modulation of antioxidant activities by low Mg [23, 24].
In vivo, Mg deficiency promotes an immuno-inflammatory response
[12, 25], which contributes to generate oxidative reactions. Since
it is well known that IL-1 and IL-6 promote oxidative stress in the
endothelium [26], it is noteworthy that Mg-deprivation abrogates
cytokine-induced ROS formation in HUVEC cultured in low Mg,
indicating that low Mg impairs the response to a receptor-mediated
stimulus leading to oxidative reactions, among others.
Conclusion
Based on our results, it is tempting to speculate that the
pro-oxidant effect of low Mg must be evaluated in a more complex
context of different contributing factors.
Further studies are required to clarify whether low Mg-induced
oxidative stress contributes to endothelial dysfunction.
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