Alteration of signaling pathways in endothelial cells by calcium phosphate bions
A.G. Kutikhin, D.K. Shishkova, E.A. Velikanova, A.V. Ponasenko
Federal State Budgetary Research Institution В«Scientific-research Institute for Complex Problems of cardiovascular disease», 650002, Kemerovo, Sosnovy boul., 6
Keywords: бионы, фосфат кальция, эндотелиальные клетки, эндотелий, атеросклероз, апоптоз, сигнальные пути, bions, calcium phosphate, endothelial cells, endothelium, atherosclerosis, apoptosis, signaling pathways
Abstract
Aim. To identify death subroutine of endothelial cells upon the exposure to calcium phosphate bions (CPB) and to define whether CPB induce specific molecular response. Materials and Methods. Immortalized murine lymphatic endothelial cells (2H-11, 85-90 % confluence) were exposed to either magnesium phosphate bions (MPB), spherical calcium phosphate bions (SCPB), or needle-shaped calcium phosphate bions (NCPB) for 24 hours with the subsequent protein extraction using radioimmunoprecipitation assay (RIPA) buffer followed by Western blotting to: 1) apoptosis effector proteins (cleaved caspase-3 (cCasp-3), cleaved poly (ADP-ribose) polymerase (cParp-1)); 2) intrinsic apoptosis proteins ((X-linked inhibitor of apoptosis protein (Xiap), survivin, plasminogen activator inhibitor 1 (Pai-1), HtrA2/Omi, cytochromec, p53 upregulated modulator of apoptosis (Puma)); 3) proteins relate to central signaling pathways (phosphorylated extracellular signal-regulated kinase (pErk), phosphorylated mitogen-activated proteinkinase (pMapk), phosphorylated focal adhesion kinase (pFak), phosphorylated nuclear factor kappa-light-chain-enhancer of activated Bcells(pNF-kB), Itch, and Gli). Results were assessed by chemiluminescence detection and using the standard ImageJ algorithm for analysis of chemiluminescent gels. Results. We found increased levels of cleaved effector caspase 3 and its cleaved substrate, cPARP-1, in endothelial cells exposed to SCPB or NCPB as compared to those exposed to MPB or control phosphate buffered saline. Further, we documented decreased level of Xiap, a key inhibitor of intrinsic apoptosis, in endothelial cells exposed to SCPB or NCPB. In contrast, survivin, an other major inhibitor of intrinsic apoptosis, and anti-apoptotic protein Pai-1 were abated upon the SCPB or NCPB exposure, possibly due to the activation of negative feedback loop. In addition, we did not detect any significant changes in master regulators of cell signaling pathways after exposure to CPB. Conclusion. CPB induce intrinsic apoptosis in endothelial cells but do not cause any specific molecular signaling response.
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