High Magnesium and Sirolimus on Rabbit Vascular Cells-An In Vitro Proof of Concept

Abstract

Drug-eluting bioresorbable scaffolds represent the last frontier in the field of angioplasty and stenting to treat coronary artery disease, one of the leading causes of morbidity and mortality worldwide. In particular, sirolimus-eluting magnesium-based scaffolds were recently introduced in clinical practice. Magnesium alloys are biocompatible and dissolve in body fluids, thus determining high concentrations of magnesium in the local microenvironment. Since magnesium regulates cell growth, we asked whether high levels of magnesium might interfere with the antiproliferative action of sirolimus. We performed in vitro experiments on rabbit coronary artery endothelial and smooth muscle cells (rCAEC and rSMC, respectively). The cells were treated with sirolimus in the presence of different concentrations of extracellular magnesium. Sirolimus inhibits rCAEC proliferation only in physiological concentrations of magnesium, while high concentrations prevent this effect. On the contrary, high extracellular magnesium does not rescue rSMC growth arrest by sirolimus and accentuates the inhibitory effect of the drug on cell migration. Importantly, sirolimus and magnesium do not impair rSMC response to nitric oxide. If translated into a clinical setting, these results suggest that, in the presence of sirolimus, local increases of magnesium concentration maintain normal endothelial proliferative capacity and function without affecting rSMC growth inhibition and response to vasodilators.

Keywords: magnesium; rabbit coronary artery endothelial cells; sirolimus; smooth muscle cells.

Figure 1

The effects of sirolimus and Mg on rCAEC. (A) rCAEC were counted after 72 and 144 h of culture in medium containing 1, 1.5, 2 or 3 mM Mg with or without different concentrations of sirolimus (SIR, 2, 10, 50 ng/mL). -SIR refers to untreated controls. (B) Confluent rCAEC were cultured in the presence of 1 or 3 mM Mg combined with 2 or 50 ng/mL of sirolimus for 48 h. Then wound assay was performed. 24 h later the width of the wound was visualized and measured. Representative images of the scratches are shown (left panel). Scale bar: 125 μm. The migration rate was analyzed using ImageJ and data are shown as the percentage of migration compared to the control (CTR, 1 mM Mg without sirolimus, right panel). (C) Confluent rCAEC were cultured in culture medium containing 1 or 3 mM Mg with or without sirolimus (2 and 50 ng/mL), stained with phalloidin and DAPI and visualized by fluorescence microscopy. Representative images are shown. Scale bar: 20 μm. Data are expressed as the mean ± standard deviation of three independent assays (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001).

Figure 2

The effects of sirolimus and Mg on the total amounts of P-selectin and VCAM. ELISA was performed on cell extracts after 72 and 144 h of culture in medium containing 1 or 3 mM Mg in the presence of 2 or 50 ng/mL of sirolimus. (A) P-selectin (B) VCAM. Data are expressed as the mean ± standard deviation of three independent assays (* p ≤ 0.05; ** p ≤ 0.01).

Figure 3

The effects of sirolimus and Mg on the total amounts of tF and eNOS. ELISA for tF (A) and eNOS (B) was performed as described above. Data are expressed as the mean ± standard deviation of three independent assays (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

Figure 4

The effects of sirolimus and Mg on rSMC. (A) The cells were counted after 72 and 144 h of culture in medium containing 1, 1.5, 2 or 3 mM Mg with or without different concentrations of sirolimus (2, 10, 50 ng/mL). (B) Confluent rSMC were cultured in the presence of 1 or 3 mM Mg combined with 2 or 50 ng/mL of sirolimus for 72 h and the wound assay was performed. 24 h later, the width of the wound was visualized and measured. Representative images of the scratches are shown (left panel). Scale bar: 125 μm. The migration rate was analyzed using ImageJ and data are shown as the percentage of migration compared to the control (CTR, 1 mM Mg without sirolimus, right panel). Data are expressed as the mean ± standard deviation of three independent assays (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001).

Figure 5

The effects of sirolimus and Mg on the response of rSMC to SNP. The cells were cultured in medium containing 1 or 3 mM Mg with or without sirolimus (2 and 50 ng/mL). After 72 h, the cells were treated with SNP for 30 min. cGMP was measured as described in the methods. Data are expressed as the mean ± standard deviation of three independent assays.