Alteration of signaling pathways in endothelial cells by calcium phosphate bions

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.

bions, calcium phosphate, endothelial cells, endothelium, atherosclerosis, apoptosis, signaling pathways

1. Heiss A., Pipich V., Jahnen-Dechent W., Schwahn D. Fetuin-A is a mineral carrier protein: small angle neutron scattering provides new insight on Fetuin-A controlled calcification inhibition // Biophys. J. 2010. Vol. 99, N 12. P. 3986-3995.

2. Wu C.Y., Young L., Young D. et al. Bions: a family of biomimetic mineralo-organic complexes derived from biological fluids // PLoS One. 2013. Vol. 8, N 9. P. e75501. doi: 10.1371/journal.pone.0075501.

3. Kutikhin A.G., Velikanova E.A., Mukhamadiyarov R.A. et al. Apoptosis-mediated endothelial toxicity but not direct calcification or functional changes in anti-calcification proteins defines pathogenic effects of calcium phosphate bions // Sci. Rep. 2016. Vol. 6. P. 27255. doi: 10.1038/srep27255.

4. Pruijm M., Lu Y., Megdiche F. et al. Serum calcification propensity is associated with renal tissue oxygenation and resistive index in patients with arterial hypertension or chronic kidney disease // J. Hypertens. 2017. Vol. 35, N 10. P. 2044-2052.

5. Smith E.R., Ford M.L., Tomlinson L.A. et al. Serum calcification propensity predicts all-cause mortality in predialysis CKD // J. Am. Soc. Nephrol. 2014. Vol. 25, N 2. P. 339-348.

6. Pasch A., Block G.A., Bachtler M. et al. Blood Calcification Propensity, Cardiovascular Events, and Survival in Patients Receiving Hemodialysis in the EVOLVE Trial // Clin. J. Am. Soc. Nephrol. 2017. Vol. 12, N 2. P. 315-322.

7. Keyzer C.A., de Borst M.H., van den Berg E. et al. Calcification Propensity and Survival among Renal Transplant Recipients // J. Am. Soc. Nephrol. 2016. Vol. 27, N 1. P. 239-248.

8. Dahle D.O., Åsberg A., Hartmann A. et al. Serum Calcification Propensity Is a Strong and Independent Determinant of Cardiac and All-Cause Mortality in Kidney Transplant Recipients // Am. J. Transplant. 2016. Vol. 16, N 1. P. 204-212.

9. O’Connell K.A., Edidin M. A mouse lymphoid endothelial cell line immortalized by simian virus 40 binds lymphocytes and retains functional characteristics of normal endothelial cells // J. Immunol. 1990. Vol. 144, N 2. P. 521-525.

10. Galluzzi L., Vitale I., Aaronson S.A. et al. Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018 // Cell Death Differ. 2018. Vol. 25, N 3. P. 486-541.

11. Green D.R., Oguin T.H., Martinez J. The clearance of dying cells: table for two // Cell Death Differ. 2016. Vol. 23, N 6. P. 915-926.

12. Morris G., Walker A.J., Berk M. et al. Cell Death Pathways: a Novel Therapeutic Approach for Neuroscientists // Mol. Neurobiol. 2017 Oct 19. doi: 10.1007/s12035-017-0793-y. [Epub ahead of print].

13. Holcik M., Korneluk R.G. XIAP, the guardian angel // Nat. Rev. Mol. Cell Biol. 2001. Vol. 2, N 7. P. 550-556.

14. Van de Walle L., Lamkanfi M., Vandenabeele P. The mitochondrial serine protease HtrA2/Omi: an overview // Cell Death Differ. 2008. Vol. 15, N 3. P. 453-460.

15. Tait S.W., Green D.R. Mitochondria and cell death: outer membrane permeabilization and beyond // Nat. Rev. Mol. Cell Biol. 2010. Vol. 11, N 9. P. 621-632.

16. Herrmann M., Schäfer C., Heiss A. et al. Clearance of fetuin-A-containing calciprotein particles is mediated by scavenger receptor-A // Circ. Res. 2012. Vol. 111, N 5. P. 575-584.

17. Kolch W. Coordinating ERK/MAPK signalling through scaffolds and inhibitors // Nat. Rev. Mol. Cell Biol. 2005. Vol. 6, N 11. P. 827-837.

18. Mitra S.K., Hanson D.A., Schlaepfer D.D. Focal adhesion kinase: in command and control of cell motility // Nat. Rev. Mol. Cell Biol. 2005. Vol. 6, N 1. P. 56-68.

19. Oeckinghaus A., Hayden M.S., Ghosh S. Crosstalk in NF-kB signaling pathways // Nat. Immunol. 2011. Vol. 12, N 8. P. 695-708.

20. Aki D., Zhang W., Liu Y.C. The E3 ligase Itch in immune regulation and beyond // Immunol. Rev. 2015. Vol. 266, N 1. P. 6-26.