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

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.

Role of L-type calcium channels and PKC in active tone development in rabbit coronary artery

The present study investigated active tone development in isolated ring segments of rabbit epicardial coronary artery. Endothelium-denuded (E−) or endothelium-intact (E+) vessels treated with the NO synthase inhibitor Nω-nitro-l-arginine (100 μM) developed active tone, which was enhanced by stretch and reversed by the NO donor sodium nitroprusside (SNP; IC50 = 9 nM). Nifedipine abolished active tone and the contractile response to phorbol dibutyrate (PDBu; 10 nM) with the same potency (IC50 = 8 nM), whereas 300 nM PDBu responses were only partially blocked by nifedipine. The classical and novel PKC inhibitors GF-109203X (IC50 = 1–2 μM) and chelerythrine (IC50 = 4–5 μM) and the classical PKC inhibitor Gö-6976 (IC50 = 0.3–0.4 μM) blocked both active tone and 10 nM PDBu responses with similar potency. Active tone development was associated with depolarization of membrane potential ( Em) and a shift to the left of the Em-vs.-contraction relationship determined by varying extracellular potassium. The depolarization and leftward shift were reversed by either chelerythrine (10 μM) or SNP (30 nM). PDBu (100–300 nM) increased peak L-type calcium channel (Cav) currents in isolated coronary myocytes, and this effect was reversed by chelerythrine (1 μM) or Gö-6976 (200 nM). SNP (500 nM) reduced Cav currents only in the presence of the PKA blocker 8-bromo-2′- O-monobutyryl-cAMPS, Rp isomer (10 μM). In conclusion, active tone development in coronary artery is suppressed by basal NO release and is dependent on both enhanced Cav activity and classical PKC activity. Both Em-dependent and -independent processes contribute to contraction. Our results suggest that one Em-independent process is direct enhancement of Cav current by PKC.