NLRP3 inhibition protects human coronary endothelial cells from oxidative and lipotoxic stress

An increased production of reactive oxygen species and elevated levels of free fatty acids are linked to inflammation and vascular endothelial dysfunction. Here, we investigated the effects of two pharmacological inhibitors of the NLRP3 inflammasome, INF150 and INF195, on Human Coronary Artery Endothelial Cells (HCAECs) exposed to hydrogen peroxide (H2O2) and palmitic acid (PA), to mimic the oxidative and lipotoxic stress underlying endothelial dysfunction. HCAECs were pre-treated for 15 min with either INF150 or INF195, followed by exposure to increasing concentrations of H2O2 or PA staring from doses that induce endothelial dysfunction without causing cell death. Cell viability was assessed by MTT assay, while in vitro angiogenesis was evaluated through pseudo-capillary formation. NLRP3, caspase-1, and IL-1β levels were quantified by ELISA and pyroptosis by LDH release and gasdermin D cleavage. Additionally, we evaluated the protective effects of the two inhibitors in HCAECs primed with TNF-α and subsequently challenged with PA. Both inhibitors preserved cell viability under oxidative stress conditions, with INF195 showing greater efficacy. Notably, only INF195 significantly protected HCAECs from both PA and TNF-α + PA-induced injury. Exposure to PA, with or without TNF-α, markedly increased caspase-1 expression and pyroptosis which were rescued by INF195. Furthermore, H2O2 and PA significantly impaired in vitro pseudocapillary formation by HCAECs and HUVECs, considered the gold standard for in vitro angiogenesis assays, an effect counteracted by INF195. These findings suggest that, under oxidative and lipotoxic stress, NLRP3 inflammasome inhibition - particularly via INF195 - supports the maintenance of vascular endothelial homeostasis by preserving cell viability and angiogenic function.