CD1 Mouse Aortic Endothelial Cells

BPA Induced Vascular Injury is Mediated by RIP3/CamKII Dependent Endothelial Cell Necroptosis

Long-term exposure to the xenoestrogen Bisphenol-A (BPA) is epidemiologically linked to adverse cardiovascular outcomes. Reventun et al. previously showed that BPA elevates blood pressure via CaMKII activation and eNOS-uncoupled oxidative stress. Since sustained CaMKII activation can trigger necroptosis, Reventun et al. tested whether BPA-induced coronary endothelial necroptosis drives cardiac dysfunction.

To investigate mechanisms underlying BPA-induced vascular leakage and myocardial hemorrhages, TUNEL assays were performed on hearts from 8-week BPA-treated CD1 mice. Total TUNEL-positive cells increased 2.8-fold, with a 9.9-fold increase in coronary vessel areas, indicating selective vascular susceptibility (Fig. 1A). However, western blot showed no changes in caspase-3 or caspase-8 activity (PARP-1 cleavage; Fig. 1B), and confocal microscopy confirmed minimal active caspase-3 staining (Fig. 1C), suggesting apoptosis-independent cell death. This was validated in vitro: BPA reduced mouse aortic endothelial cell (MAEC) viability from 10^-7 M (Fig. 2A) without activating caspase-3 or -8 at 10^-6 M (Fig. 2B), whereas cardiomyocytes (H9c2) remained viable except at 10^-4 M (Fig. 2C), confirming endothelial-specific, non-apoptotic death. Since caspase-8 negatively regulates necroptosis by cleaving RIP1/3, we examined RIP3 expression. RIP3 was upregulated in BPA-treated hearts at 4, 8, and 16 weeks (Fig. 1D), with intense endothelial localization at 8 weeks (Fig. 1E). Cell fractionation revealed specific RIP3 increase in CD31^+ endothelial cells but not cardiomyocytes (Fig. 1F), and H9c2 cells showed no RIP3 changes (Fig. 2D), indicating BPA activates RIP3-mediated necroptosis selectively in vascular cells.