LYSOSOME-DEPENDENT CELL DEATH DEFINES SPECIFIC ENDOTHELIAL TOXICITY OF CALCIUM PHOSPHATE BIONS
Daria Kirillovna SHISHKOVA1, Elena Anatol’evna VELIKANOVA1, Rinat Avkhadievich MUKHAMADIYAROV1, Arseniy Evgen’evich YUZHALIN1, Yuliya Aleksandrovna KUDRYAVTSEVA1, Anna Nikolaevna POPOVA2, Dmitriy Mikhaylovich RUSSAKOV3, Anton Gennad’evich KUTIKHIN1
1Research Institute for Complex Issues of Cardiovascular Diseases 2Institute of Coal Сhemistry and Material Science 3Kemerovo State University
Keywords: бионы, фосфат кальция, гидроксиапатит, токсичность, эндотелий, лизосомы, клеточная гибель, атеросклероз, bions, calcium phosphate, hydroxyapatite, toxicity, endothelium, lysosomes, cell death, atherosclerosis
Abstract
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.
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