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Magnesium deficiency and endothelial dysfunction: is oxidative stress involved?


Magnesium Research. Volume 21, Number 1, 58-64, march 2008, original article

DOI : 10.1684/mrh.2008.0129

Summary  

Author(s) : Federica I Wolf, Valentina Trapani, Matteo Simonacci, Silvia Ferré, Jeanette AM Maier , Istituto di Patologia Generale, e Centro di Ricerche Oncologiche Giovanni XXIII, Facoltà di Medicina, Università Cattolica del Sacro Cuore, Roma, Dipartimento di Scienze Precliniche LITA Vialba, Università di Milano, Milano, Italy.

Summary : Low magnesium (Mg) has been associated with oxidative stress, an important player in aging, atherosclerosis and other vascular diseases. In vivo, low Mg and immune system activation seem to cooperate to promote endothelial dysfunction. We therefore evaluated whether exposure of human endothelial cells to low Mg in vitro determines oxidative stress features. We therefore measured intracellular reactive oxygen species (ROS) by dichlorofluorescein (DCF) fluorescence after Mg deprivation with or without treatment with H2O2. While we did not observe any alteration of DCF-detectable intracellular ROS under basal conditions, we show that, early after exposure to low Mg (2 h), endothelial cells are more sensitive to the oxidant action of H2O2 than the controls cultured in physiologic concentrations of Mg. This increase of ROS in Mg deprived cells is transient and followed by a stable reduction of DCF-fluorescence below the levels measured in the controls. We also evaluated oxidative DNA damage and observed higher 8-hydroxy-deoxyguanine levels early (2 h) after Mg deprivation in respect to the controls, both in basal conditions and after treatment with H2O2. Mg deficiency in vivo associates with the onset of an inflammatory response leading to increased circulating levels of cytokines, which trigger an oxidative response in endothelial cells. We here show that exposure to IL-1 and IL-6 significantly increased the levels of DCF-detectable ROS in cells cultured in physiologic concentrations of Mg, but not in Mg-deprived cells. We conclude that low Mg transiently leads to pro-oxidant effects. We suggest that different molecules, including pro-inflammatory cytokines, might be involved in promoting endothelial dysfunction.

Keywords : magnesium, oxidative stress, endothelial cells, 8-OHdG

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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.

References

1 Cines DB, Pollak ES, Buck CA, Loscalzo J, Zimmerman GA, McEver RP, Pober JS, Wick TM, Konkle BA, Schwartz BS, Barnathan ES, McCrae KR, Hug BA, Schmidt AM, Stern DM. Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood 1998; 91: 3527-61.

2 Ross R. Atherosclerosis – an inflammatory disease. N Engl J Med 1999; 340: 115-26.

3 Lusis AJ. Atherosclerosis. Nature 2000; 407: 233-41.

4 Maier JAM. Low magnesium and atherosclerosis: an evidence-based link. Mol Aspects Med 2003; 24: 137-46.

5 Yang ZW, Gebrewold A, Nowakowski M, Altura BT, Altura BM. Mg(2+)-induced endothelium-dependent relaxation of blood vessels and blood pressure lowering: role of NO. Am J Physiol Regul Integr Comp Physiol 2000; 278: R628-R639.

6 Yokoyama S, Gu J, Nishida HI, Smith TL, Kummerow FA. Combined effects of magnesium deficiency and an atherogenic level of low density lipoprotein on uptake and metabolism of low density lipoprotein by cultured human endothelial cells. II. Electron microscopic data. Magnes Res 1994; 7: 97-105.

7 Maier JAM, Malpuech-Brugère C, Zimowska W, Rayssiguier Y, Mazur A. Low magnesium promotes endothelial cell dysfunction: implications for atherosclerosis, inflammation and thrombosis. Biochim Biophys Acta 2004; 1689: 13-21.

8 Ferré S, Mazur A, Maier JAM. Low-magnesium induces senescent features in cultured human endothelial cells. Magnes Res 2007; 20: 66-71.

9 Bonomini F, Tengattini S, Fabiano A, Bianchi R, Rezzani R. Atherosclerosis and oxidative stress. Histol Histopathol 2008; 23: 381-90.

10 Donato AJ, Eskurza I, Silver AE, Levy AS, Pierce GL, Gates PE, Seals DR. Direct evidence of endothelial oxidative stress with aging in humans: relation to impaired endothelium-dependent dilation and upregulation of nuclear factor-kappaB. Circ Res 2007; 100: 1659-66.

11 Maier JAM, Nasulewicz-Goldeman A, Simonacci M, Boninsegna A, Mazur A, Wolf FI. Insights into the mechanisms involved in magnesium-dependent inhibition of primary tumor growth. Nutr Cancer 2007; 59: 192-8.

12 Malpuech-Brugere C, Nowacki W, Daveau M, Gueux E, Linard C, Rock E, Lebreton J, Mazur A, Rayssiguier Y. Inflammatory response following acute magnesium deficiency in the rat. Biochim Biophys Acta 2000; 1501: 91-8.

13 Manju L, Nair RR. Magnesium deficiency augments myocardial response to reactive oxygen species. Can J Physiol Pharmacol 2006; 84: 617-24.

14 Yang Y, Wu Z, Chen Y, Qiao J, Gao M, Yuan J, Nie W, Guo Y. Magnesium deficiency enhances hydrogen peroxide production and oxidative damage in chick embryo hepatocyte in vitro. Biometals 2006; 19: 71-81.

15 Hartwig A. Role of magnesium in genomic stability. Mutat Res 2001; 475: 113-21.

16 Wiles ME, Wagner TL, Weglicki WB. Effect of acute magnesium deficiency (MgD) on aortic endothelial cell (EC) oxidant production. Life Sci 1997; 60: 221-36.

17 Yang Y, Wu Z, Chen Y, Qiao J, Gao M, Yuan J, Nie W, Guo Y. Magnesium deficiency enhances hydrogen peroxide production and oxidative damage in chick embryo hepatocyte in vitro. Biometals 2006; 19: 71-81.

18 Manju L, Nair RR. Magnesium deficiency augments myocardial response to reactive oxygen species. Can J Physiol Pharmacol 2006; 84: 617-24.

19 Dickens BF, Weglicki WB, Li YS, Mak IT. Magnesium deficiency in vitro enhances free radical-induced intracellular oxidation and cytotoxicity in endothelial cells. FEBS Lett 1992; 311: 187-91.

20 Freedman AM, Mak IT, Stafford RE, Dickens BF, Cassidy MM, Muesing RA, Weglicki WB. Erythrocytes from magnesium-deficient hamsters display an enhanced susceptibility to oxidative stress. Am J Physiol 1992; 262: C1371-C1375.

21 Martin H, Richert L, Berthelot A. Magnesium deficiency induces apoptosis in primary cultures of rat hepatocytes. J Nutr 2003; 133: 2505-11.

22 Astier C, Rock E, Lab C, Gueux E, Mazur A, Rayssiguier Y. Functional alterations in sarcoplasmic reticulum membranes of magnesium-deficient rat skeletal muscle as consequences of free radical-mediated process. Free Radic Biol Med 1996; 20: 667-74.

23 Vernet P, Britan A, Gueux E, Mazur A, Drevet JR. Dietary magnesium depletion does not promote oxidative stress but targets apical cells within the mouse caput epididymidis. Biochim Biophys Acta 2004; 1675: 32-45.

24 Zhou Q, Olinescu RM, Kummerow FA. Influence of low magnesium concentrations in the medium on the antioxidant system in cultured human arterial endothelial cells. Magnes Res 1999; 12: 19-29.

25 Rayssiguier Y, Gueux E, Nowacki W, Rock E, Mazur A. High fructose consumption combined with low dietary magnesium intake may increase the incidence of the metabolic syndrome by inducing inflammation. Magnes Res 2006; 19: 237-43.

26 Kofler S, Nickel T, Weis M. Role of cytokines in cardiovascular diseases: a focus on endothelial responses to inflammation. Clin Sci 2005; 108: 205-13.


 

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