PATHOLOGICAL EFFECTS OF IONIZED CALCIUM, CALCIPROTEIN MONOMERS AND CALCIPROTEIN PARTICLES ON ARTERIAL ENDOTHELIAL CELLS

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Abstract
Highlights To achieve a 10% (i.e., 1.1-fold) increase in ionized calcium level in the serum-free cell culture medium and Wistar rat serum, 10 µg/mL calcium (e.g. with CaCl2) should be added; this corresponds to the upper quartile of ionized calcium in the human population. Incubation with ionized calcium or calciprotein monomers (10 µg/mL calcium) does not induce any pathological effects in primary arterial endothelial cells, although calciprotein monomers are internalised by endothelial cells similar to calciprotein particles. Incubation of primary human coronary artery endothelial cells with calciprotein particles (10 µg/mL calcium) triggers an increased expression of VCAM1, ICAM1, and SELE genes (i.e., those encoding cell adhesion molecules) as well as IL6, CXCL8, and CXCL1 genes (i.e., those encoding pro-inflammatory cytokines), together indicating endothelial activation. Aim. To compare the pathological effects of ionized calcium, calciprotein monomers (CPMs), and calciprotein particles (CPPs) after their addition to endothelial cells.Methods. CPMs and CPPs were synthesized by the supersaturation of albumin-supplemented NaCl solution with calcium (by adding CaCl2) and phosphate ions (by adding Na2HPO4). CPMs and CPPs were separated by sequential ultracentrifugation (to isolate CPPs) and ultrafiltration (to separate CPMs and free mineral ions). Calcium concentration in CPMs and CPPs was measured by an o-cresolphthalein complexone-based colorimetric assay. Internalization of fluorescent-labeled CPMs and CPPs by endothelial cells was interrogated by confocal microscopy after their 1-hour co-incubation under flow. Primary coronary artery and internal thoracic artery endothelial cells were incubated with ionized calcium (CaCl2), CPMs, or CPPs (10 µg/mL calcium) upon reaching confluence (n = 6 wells per group) to simulate a 10% increase in ionized calcium concentration. After 24 hours, we collected RNA and analyzed the gene expression pattern by reverse transcription-quantitative polymerase chain reaction.Results. CPMs and CPPs were internalized by endothelial cells as soon as after 1 hour of co-incubation under flow. Treatment with ionized calcium or CPMs (10 µg/mL calcium) did not cause any pathological effects in arterial endothelial cells. In contrast, CPPs promoted an expression of VCAM1, ICAM1, and SELE genes (i.e., those encoding cell adhesion molecules) as well as IL6, CXCL8, and CXCL1 genes (i.e., those encoding pro-inflammatory cytokines) in primary human coronary artery endothelial cells, together indicating their pro-inflammatory activation.Conclusion. Pathological effects of calcium stress are defined by the calcium source and not simply by its concentration.

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