Endothelial cell death subroutines at physiological and supraphysiological concentrations of calcium phosphate bions

Abstract

Background. Calcium phosphate bions (CPB) formed and circulating in the blood at its supersaturation with calcium and phosphate provoke endothelial dysfunction by causing the demise of arterial endothelial cells (ECs). Aim. To examine cell death subroutines of human primary arterial ECs exposed to physiological and supraphysiological concentrations of spherical CPB (CPB-S) and needle-shaped CPB (CPB-N). Materials and methods. Equal volumes (10 μL) of CPB-S and CPB-N at physiological concentration (optical density at 650 nm wavelength = 0.08-0.10) or supraphysiological amounts (optical density at 650 nm wavelength = 0.42-0.45) were added to the confluent primary human coronary artery and internal thoracic artery ECs cultured in 96-well plates. In all experimental groups, we selectively inhibited caspase-3 by adding Z-D(OMe)E(OMe)VD(OMe)-FMK (100 μmol/L) or
all caspases (Z-VAD(OMe)-FMK, 100 μmol/L) to assess whether the CPB-induced cell death is regulated or accidental. Cell viabilitywas evaluated by sequential colorimetric determination of metabolic activity at 4, 24, and 48 hours of incubation with CPB. Results. At supraphysiological CPB concentrations, the majority (60-85%) of ECs died regardless of CPB type and caspase inhibitors, albeit at 24- and 48-hour time points the latter had minor cytoprotective action. However at physiological CPB levels, caspase inhibitors rescued a considerable proportion of ECs after 24 or 48 hours of exposure, and CPB-N had significantly higher toxicity than CPB-S. Regardless of the time point, the cytoprotective effect of the pan-caspase inhibitor was significantly higher than that of the selective caspase-3 inhibitor indicating a cumulative caspase inhibition and suggesting that cell death was precipitated by an intrinsic apoptosis pathway. Conclusion. At supraphysiological concentrations, CPB cause instant cell death; yet at physiological amounts, caspase inhibitors rescue the majority of ECs testifying to the regulated cell death. Further studies in this field should decipher the molecular pathways of CPB-induced regulated cell death of ECs.