Axitinib and pembrolizumab impaired endothelial barrier integrity and increased oxidative stress in human aortic endothelial cells, effects that were attenuated by NOX1/4 inhibition (p<0.05).
Axitinib and pembrolizumab induce endothelial dysfunction through NOX-dependent oxidative stress, highlighting NOX inhibition as a potential strategy to mitigate anti-cancer therapy-associated vascular toxicity.
p-value: p=<0.05
Objective: Vascular toxicity is increasingly recognised with vascular endothelial growth factor inhibitor (VEGFi) and immune checkpoint inhibitor (ICI) cancer therapies, particularly when used in combination; however, the mechanisms underlying endothelial dysfunction remain poorly understood. This study investigated whether oxidative stress mediated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) 1 and 4 contributes to endothelial dysfunction induced by axitinib (VEGFi) and pembrolizumab (ICI) in human aortic endothelial cells (HAECs). Design and method: Primary HAECs were exposed to axitinib (1 μM) and pembrolizumab (5 μg/ml) alone and in combination. In vitro studies evaluated endothelial barrier integrity using transendothelial electrical resistance (TEER) measurements (day 0, 2, 4, 7 and 10) and immunofluorescent analysis of junctional proteins (48 hours). Oxidative stress (24 hours) was evaluated by measuring intracellular reactive oxygen species (ROS), mitochondrial superoxide, and oxidative DNA damage by immunofluorescence. Involvement of NOXs was examined using dual pharmacological inhibition of NOX1/4 (GKT137831) and selective inhibition of NOX1 (ML171) and NOX4 (GLX351322). Additional assessments included RT-qPCR and extracellular vesicles (EV) release. Results: Axitinib and pembrolizumab impaired endothelial barrier integrity, demonstrated by a reduced TEER value at days 4, 7 and 10 and decreased expression of vascular endothelial cadherin and zonula occludens-1 (p<0.05). Pre-treatment with GKT137831 attenuated this barrier dysfunction, partially restoring TEER (p<0.05). Increased expression of DNA lesion marker, 8-hydroxy-2’-deoxyguanosine (8-OHdG), indicative of oxidative stress, was observed in HAECs treated with monotherapy and combination therapy (p<0.05). Mitochondrial-specific superoxide production was also increased in HAECs treated with axitinib and pembrolizumab (p<0.05). Ongoing studies are investigating whether these oxidative stress responses are dependent on NOX1 or NOX4 using selective pharmacological inhibition. Gene expression analysis indicates increased NOX1 and reduced NOX4 expression following axitinib treatment. Axitinib and combination treatment also increased EV release in HAECs, suggesting stress-induced paracrine signalling associated with oxidative injury and redox imbalance. Conclusions: Our findings demonstrate that cancer therapies induce endothelial dysfunction through increased oxidative stress and disruption of endothelial cells barrier integrity in a NOX-dependent manner. Modulation of NOX redox signalling may represent a potential strategy to mitigate vascular toxicity associated with anti-cancer therapies.
Whelan et al. (2026) studied Endothelial dysfunction. Axitinib and pembrolizumab vs. Control / Monotherapy was evaluated on Endothelial barrier integrity (TEER) and oxidative stress markers (p=<0.05). Axitinib and pembrolizumab impaired endothelial barrier integrity and increased oxidative stress in human aortic endothelial cells, effects that were attenuated by NOX1/4 inhibition (p<0.05).