Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells

Cardiovascular (CV) benefits of Natural Antioxidant (NA) supplementation are contradictory. Endothelial Cells (EC) are pivotal player on CV diseases onset/progression and thus represent a good model to study the NA impact on vascular pathophysiology. We show that two NA, coumaric acid and resveratrol, affect intracellular ROS levels and cell physiology in human EC. While at lower doses both compounds were antioxidant, at mildly high doses they became pro-oxidant, eliciting cell death by apoptosis and phospho-Akt inhibition. Pro-oxidant effects were counteracted by diphenyleneiodonium, hinting a role for flavin oxidases in NA-induced toxicity. ECs treatment with the mitochondrial permeability transition pore inhibitor prevented oxidative cell damage indicating mitochondrial involvement in NA-induced ECs impairment. Pro-oxidant effects were also hindered by sulfaphenazole (SPZ), suggesting a role for CYP2C9 in NA-induced toxicity. SPZ also prevented NA-induced p-Akt down-regulation and mitochondrial membrane potential (MMP) impairment indicating that Akt works downstream of CYP2C9 in mediating cellular responses to NA. Stimulation of p-Akt by insulin, counteracted NA-induced MMP impairment and cell death, an effect abolished by Akt inhibitors further suggesting that Akt regulates cell survival in response to NA-induced stress. Our study show that mildly high-doses of NA induce mitochondria-dependent cell damage mediated by CYP2C9 and the Akt pathway in a human vascular model.