LYSOSOME-DEPENDENT CELL DEATH DEFINES SPECIFIC ENDOTHELIAL TOXICITY OF CALCIUM PHOSPHATE BIONS

Aim of the study was to identify the mechanism of specific endothelial toxicity related to calcium phosphate bions (CPB). Material and methods. CPB and magnesium phosphate bions (MPB) were artificially synthesised through supersaturation of culture medium with respective salts and then added to human endothelial cells (EA.hy 926) and murine endothelial cells (2H-11) to study: 1) spatiotemporal aspects of bion internalisation by means of transmission electron microscopy and confocal microscopy; 2) whether blocking of H+-ATPase by lysosomal inhibitor bafilomycin A1 affects endothelial toxicity of bions; 3) expression of caspase-3 and its substrate poly(ADP-ribose) polymerase (PARP-1). Results. CPB were internalized by endothelial cells as early as 1 h upon their addition and were localized in lysosomes; after 4 h, we detected release of calcium ions (Ca2+) from lysosomes to cytosol accompanied by multifold increase in cleaved caspase 3 and its substrate PARP-1. Bafilomycin A1 rescued endothelial cells from death induced by slightly soluble CPB regardless of exposure time and dose; however, freely soluble MPB did not evince endothelial toxicity regardless of bafilomycin A1 addition. Conclusion. Upon internalization by endothelial cells, CPB cause their death due to dissolution in lysosomes and subsequent release of calcium ions into the cytosol, ultimately leading to cleavage of executioner caspases. MPB lack endothelial toxicity because their dissolution does not lead to release of calcium ions. Therefore, specific endothelial toxicity of CPB is defined by lysosome-dependent cell death.

1. Kutikhin A.G., Sinitsky M.Yu., Ponasenko A.V. The role of mutagenesis in atherosclerosis. Kompleksnye problemy serdechno-sosudistykh zabolevaniy = Complex Issues of Cardiovascular Diseases. 2017; (1): 92–101. [In Russian].

2. Shishkova D.K., Velikanova E.A., Krivkina E.O., Mironov A.V., Kudryavtseva Yu.A., Kutikhin A.G. Calcium-phosphate bions do specifically induce hypertrophy of damaged intima in rats. Rossiyskiy kardiologicheskiy zhurnal = Russian Journal of Cardiology. 2018; 23 (9): 33–38. [In Russian].

3. Shishkova D.K., Velikanova E.A., Krivkina E.O., Mironov A.V., Kudryavtseva Yu.A., Kutikhin A.G. Toxicity of calcium phosphate bions for aortic adventitia in rats. Ateroskleroz i dislipidemii = The Journal of Atherosclerosis and Dyslipidemias. 2018; 3: 37–43. [In Russian].

4. Bowman E.J., Siebers A., Altendorf K. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc. Natl. Acad. Sci. USA. 1988; 85 (21): 7972–7976.

5. Ewence A.E., Bootman M., Roderick H.L., Skepper J.N., McCarthy G., Epple M., Neumann M., Shanahan C.M., Proudfoot D. Calcium phosphate crystals induce cell death in human vascular smooth muscle cells: a potential mechanism in atherosclerotic plaque destabilization. Circ. Res. 2008; 103 (5): e28–e34.

6. Foley R.N., Collins A.J., Ishani A., Kalra P.A. Calcium-phosphate levels and cardiovascular disease in community-dwelling adults: the Atherosclerosis Risk in Communities (ARIC) Study. Am. Heart J. 2008; 156 (3): 556–563.

7.

8. Julien O., Wells J.A. Caspases and their substrates. Cell Death Differ. 2017; 24 (8): 1380–1389.

9. Kumar S., Kasseckert S., Kostin S., Abdallah Y., Schafer C., Kaminski A., Reusch H.P., Piper H.M., Steinhoff G., Ladilov Y. Ischemic acidosis causes apoptosis in coronary endothelial cells through activation of caspase-12. Cardiovasc. Res. 2007; 73 (1): 172–180.

10. Kutikhin A.G., Velikanova E.A., Mukhamadiyarov R.A., Glushkova T.V., Borisov V.V., Matveeva V.G., Antonova L.V., Filipev D.E., Golovkin A.S., Shishkova D.K., Burago A.Y., Frolov A.V., Dolgov V.Y., Efimova O.S., Popova A.N., Malysheva V.Y., Vladimirov A.A., Sozinov S.A., Ismagilov Z.R., Russakov D.M., Lomzov A.A., Pyshnyi D.V., Gutakovsky A.K., Zhivodkov Y.A., Demidov E.A., Peltek S.E., Dolganyuk V.F., Babich O.O., Grigoriev E.V., Brusina E.B., Barbarash O.L., Yuzhalin A.E. Apoptosismediated endothelial toxicity but not direct calcification or functional changes in anti-calcification proteins defines pathogenic effects of calcium phosphate bions. Sci. Rep. 2016; 6: 27255.

11. Larsson T.E., Olauson H., Hagström E., Ingelsson E., Arnlöv J., Lind L., Sundström J. Conjoint effects of serum calcium and phosphate on risk of total, cardiovascular, and noncardiovascular mortality in the community. Arterioscler. Thromb. Vasc. Biol. 2010; 30 (2): 333–339.

12. Lind L., Skarfors E., Berglund L., Lithell H., Ljunghall S. Serum calcium: a new, independent, prospective risk factor for myocardial infarction in middle-aged men followed for 18 years. J. Clin. Epidemiol. 1997; 50 (8): 967–973.

13. Liu Z., Xiao Y., Chen W., Wang Y., Wang B., Wang G., Xu X., Tang R. Calcium phosphate nanoparticles primarily induce cell necrosis through lysosomal rupture: the origination of material cytotoxicity. J. Mater. Chem. B. 2014; 2: 3480–3489.

14. Shalini S., Dorstyn L., Dawar S., Kumar S. Old, new and emerging functions of caspases. Cell Death Differ. 2015; 22 (4): 526–539.

15. Shyong Y.J., Wang M.H., Kuo L.W., Su C.F., Kuo W.T., Chang K.C., Lin F.H. Mesoporous hydroxyapatite as a carrier of olanzapine for longacting antidepression treatment in rats with induced depression. J. Control Release. 2017; 255: 62–72.

16. Shyong Y.J., Wang M.H., Tseng H.C., Cheng C., Chang K.C., Lin F.H. Mesoporous hydroxyapatite as olanzapine carrier provides a long-acting effect in antidepression treatment. J. Med. Chem. 2015; 58 (21): 8463–8474.

17. Tewari M., Quan L.T., O’Rourke K., Desnoyers S., Zeng Z., Beidler D.R., Poirier G.G., Salvesen G.S., Dixit V.M. Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADPribose) polymerase. Cell. 1995; 81 (5): 801–809.

18. Wu C.Y., Young L., Young D., Martel J., Young J.D. Bions: a family of biomimetic mineraloorganic complexes derived from biological fluids. PLoS One. 2013; 8 (9): e75501.

19. Yoshimori T., Yamamoto A., Moriyama Y., Futai M., Tashiro Y. Bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J. Biol. Chem. 1991; 266 (26): 17707–17712.

20. Yuan Y., Liu C., Qian J., Wang J., Zhang Y. Sizemediated cytotoxicity and apoptosis of hydroxyapatite nanoparticles in human hepatoma HepG2 cells. Biomaterials. 2010; 31 (4): 730–740.

21. Zhang C.Y., Sun X.Y., Ouyang J.M., Gui B.S. Diethyl citrate and sodium citrate reduce the cytotoxic effects of nanosized hydroxyapatite crystals on mouse vascular smooth muscle cells. Int. J. Nanomedicine. 2017; 12: 8511–8525.