Magnesium Deficiency Induces Lipid Accumulation in Vascular Endothelial Cells via Oxidative Stress-The Potential Contribution of EDF-1 and PPARγ

Abstract

Background: Magnesium deficiency contributes to atherogenesis partly by promoting the dysfunction of endothelial cells, which are critical in vascular homeostasis and diseases. Since EDF-1 and PPARγ regulate crucial endothelial activities, we investigated the modulation of these proteins involved in lipogenesis as well the deposition of lipids in human endothelial cells cultured in different concentrations of magnesium.

Methods: Human endothelial cells from the umbilical vein were cultured in medium containing from 0.1 to 5 mM magnesium for 24 h. The levels of EDF-1 and PPARγ were visualized by Western blot. Reactive oxygen species (ROS) were measured by DCFDA. Lipids were detected after O Red Oil staining.

Results: Magnesium deficiency leads to the accumulation of ROS which upregulate EDF-1. Further, PPARγ is increased after culture in low magnesium, but independently from ROS. Moreover, lipids accumulate in magnesium-deficient cells.

Conclusions: Our results suggest that magnesium deficiency leads to the deposition of lipids by inducing EDF-1 and PPARγ. The increase in intracellular lipids might be interpreted as an adaptive response of endothelial cells to magnesium deficiency.

Keywords: EDF-1; PPARγ; endothelial cells; lipids; magnesium.

Figure 1

Low magnesium induces EDF-1. HUVEC were cultured in medium containing 0.1, 0.3, 0.5, 1 or 5 mM of extracellular Mg for 24 h. Cell extracts were processed for Western blot using antibodies against EDF-1. Actin was used as a control of loading. A representative blot is shown. Densitometry (right panel) was performed on three different blots using Image J. ** p ≤ 0.01.

Figure 2

Low magnesium induces ROS accumulation. ROS production was evaluated using DCFDA in HUVEC maintained for 24 h in media containing different concentrations of Mg in the presence (orange columns) or in the absence (green columns) of NAC (5 mM). Fluorescence at 529 nm was measured and then normalized to the cell number. The experiment was performed three times in triplicates. * p ≤ 0.05 and ** p ≤ 0.01.

Figure 3

Oxidative stress induces EDF-1. (A) HUVEC were pretreated or not with NAC (5 mM) for 1 h, exposed or not to H₂O₂ (200 μM) for 30 min and extracted 24 h later. (B) HUVEC were cultured in 0.1 or 1 mM of Mg in the presence or in the absence of NAC (5 mM) for 24 h. Western blot was performed using antibodies against EDF-1. Actin was used as a control of loading. A representative blot is shown. Densitometry (right panel) was performed on three different blots using Image J. * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.

Figure 4

Low magnesium induces PPARγ. (A) HUVEC were cultured in low (0.1, 0.3, 0.5 mM), physiological (1 mM) and high (5 mM) extracellular Mg for 24 h. (B) HUVEC were cultured in 0.1 or 1 mM in the presence or in the absence of NAC (5 mM) for 24 h. (C) HUVEC were treated with H₂O₂ (200 μM) for 30 min in the presence or in the absence of NAC (5 mM) and then maintained in culture for 24 h. Cell extracts were processed for Western blot using antibodies against PPARγ. Actin was used as a control of loading. A representative blot is shown. Densitometry (right panel) was performed on three different blots by Image J. * p ≤ 0.05 and ** p ≤ 0.01.

Figure 5

Low Mg induces lipid accumulation. HUVEC were cultured in different concentrations of Mg. 24 h later, the cells were stained with Oil Red O and solubilized. Absorbance was measured at 500 nm and normalized to the cell number. The experiment was performed three times in triplicates. ** p ≤ 0.01 and **** p ≤ 0.0001.