Fig. 5.

Induction of intracellular calcium overload or promotion of mitochondrial calcium entry induces endothelial cell necroptosis. Primary CMECs were cultured in a vascular-cell basal medium supplemented with the endothelial cell growth kit VEGF. Hypoxia/reoxygenation (H/R) injury was induced through 30 min of hypoxia and 2 h of reoxygenation. SERCA AAV9 or control AAV9 vectors were transfected into CMECs, which were termed SERCA^AAV9 group or control group respectively. [Ca²⁺]i activator (ionomycin, 3 μM for 2 h) and MCU agonist (spermine, 10 μM for 2 h) were administrated into SERCA-overexpressed CMECs to offset the regulatory effects of SERCA on calcium homeostasis and MCU inhibition. A. Cell death was measured using the LDH cytotoxicity assay. B. An MTT assay was used to observe the viability of CMECs in response to H/R injury. C. ATP production was measured using ELISA. D–F. Proteins were isolated from H/R-treated CMECs, and then the levels of Ripk3 and PGAM5 were measured through western blots. *p < .05.