Accepted_test

Background: Calciprotein particles (CPPs) are indispensable scavengers of excessive Ca²⁺ and PO₄^3- ions in blood, being internalised and recycled by liver and spleen macrophages, monocytes, and endothelial cells (ECs). Supraphysiological concentrations of CPPs cause endothelial cell dysfunction by inducing release of pro-inflammatory cytokines. Here we evaluated whether physiological levels of CPPs, which correspond to 10% increase in serum ionised calcium, and similar amounts of calcium incorporated into calciprotein monomers (CPM) are capable of causing endothelial activation.
Methods: CPM and CPPs were synthesised using supersaturation of saline solution with Ca²⁺ and PO₄^3- ions and albumin as a single mineral chaperone. Upon the addition of physiological calcium levels (10 μg/mL), delivered as ionised calcium (Ca²⁺), albumin-centric CPM, or albumin-centric CPPs, to human coronary artery endothelial cells (HCAEC) and human internal thoracic artery endothelial cells (HITAEC) for 24 hours, we measured gene expression by RT-qPCR and pro-inflammatory cytokines by dot blotting and ELISA.
Results: Albumin-centric CPPs and CPM were internalised by HCAEC and HITAEC after 1-hour incubation in the pulsatile flow system. In contrast to Ca2+ ions and albumin-centric CPM, albumin-centric CPPs caused a significant increase in production of IL-6, IL-8, and MCP-1/CCL2 into the milieu and elevated expression of VCAM1, ICAM1, SELE, IL6, CXCL8, and CXCL1 genes by HCAEC and HITAEC after 24 hours of incubation.
Conclusion: Among three distinct mechanisms of calcium delivery, albumin-centric CPPs were the only which provoked pro-inflammatory endothelial cell dysfunction if added at physiological levels (10 μg/mL), whilst Ca²⁺ ions and CPM did not cause significant alterations.