Molecular biology reports · 2026
Development of endothelial injury under chemical hypoxia: early migration impairment precedes cytotoxicity in CoCl2-treated EA.hy926 cells.
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Kolesnikov Andrey E, Petrova Anastasia A, Cherednichenko Vadim R, Elizova Natalia V, Gladysheva Nadezhda P, Zakharova Elena N, Kirichenko Tatiana V, Markin Alexander M
Аннотация
BACKGROUND: Cobalt(II) chloride (CoCl2) in vitro models are widely used to study hypoxia-associated endothelial dysfunction, but exposure conditions and biological endpoints remain poorly standardized, and the temporal sequence of the underlying events is largely uncharacterized. This study aimed to establish a standardized model of severe CoCl2-induced endothelial dysfunction and characterize its temporal progression.
METHODS AND RESULTS: A standardized model was established using the 24-h IC50 concentration of CoCl2 (0.70 ± 0.24 mM) in EA.hy926 cells, and stage-specific responses were assessed at 12, 24, and 48 h by flow cytometric annexin V/PI, RT-qPCR, and scratch wound-healing assays. Impairment of endothelial migratory capacity was the earliest detectable manifestation of dysfunction, evident by 12 h in the absence of significant apoptosis or major transcriptional changes. By 24 h, CoCl2 induced a significant increase in early apoptotic cells, while 48-h exposure promoted progression towards late apoptosis/secondary necrosis and further loss of viability. RT-qPCR revealed progressive downregulation of HIF-1α accompanied by upregulation of PHD2, indicating negative-feedback regulation during severe chemical hypoxia, whereas VEGF-165 and FOXO3 expression remained largely unchanged.
CONCLUSIONS: These findings identify distinct sequential stages of CoCl2-induced endothelial dysfunction, in which functional impairment of endothelial migration precedes overt cytotoxicity and apoptosis. The model provides a platform for evaluating endothelial-protective therapeutic strategies. However, as EA.hy926 is a hybrid cell line, these findings should be confirmed in primary endothelial cells before clinical extrapolation.
METHODS AND RESULTS: A standardized model was established using the 24-h IC50 concentration of CoCl2 (0.70 ± 0.24 mM) in EA.hy926 cells, and stage-specific responses were assessed at 12, 24, and 48 h by flow cytometric annexin V/PI, RT-qPCR, and scratch wound-healing assays. Impairment of endothelial migratory capacity was the earliest detectable manifestation of dysfunction, evident by 12 h in the absence of significant apoptosis or major transcriptional changes. By 24 h, CoCl2 induced a significant increase in early apoptotic cells, while 48-h exposure promoted progression towards late apoptosis/secondary necrosis and further loss of viability. RT-qPCR revealed progressive downregulation of HIF-1α accompanied by upregulation of PHD2, indicating negative-feedback regulation during severe chemical hypoxia, whereas VEGF-165 and FOXO3 expression remained largely unchanged.
CONCLUSIONS: These findings identify distinct sequential stages of CoCl2-induced endothelial dysfunction, in which functional impairment of endothelial migration precedes overt cytotoxicity and apoptosis. The model provides a platform for evaluating endothelial-protective therapeutic strategies. However, as EA.hy926 is a hybrid cell line, these findings should be confirmed in primary endothelial cells before clinical extrapolation.