Empagliflozin prevents oxidative stress in human coronary artery endothelial cells via the NHE/PKC/NOX axis

Abstract

Background: Empagliflozin (EMPA) ameliorates reactive oxygen species (ROS) generation in human endothelial cells (ECs) exposed to 10 % stretch, but the underlying mechanisms are still unclear. Pathological stretch is supposed to stimulate protein kinase C (PKC) by increasing intracellular calcium (Ca²⁺), therefore activating nicotinamide adenine dinucleotide phosphate oxidase (NOX) and promoting ROS production in human ECs. We hypothesized that EMPA inhibits stretch-induced NOX activation and ROS generation through preventing PKC activation.

Methods: Human coronary artery endothelial cells (HCAECs) were pre-incubated for 2 h before exposure to cyclic stretch (5 % or 10 %) with either vehicle, EMPA or the PKC inhibitor LY-333531 or PKC siRNA. PKC activity, NOX activity and ROS production were detected after 24 h. Furthermore, the Ca²⁺ chelator BAPTA-AM, NCX inhibitor ORM-10962 or NCX siRNA, sodium/potassium pump inhibitor ouabain and sodium hydrogen exchanger (NHE) inhibitor cariporide were applied to explore the involvement of the NHE/Na⁺/NCX/Ca²⁺ in the ROS inhibitory capacity of EMPA.

Results: Compared to 5 % stretch, 10 % significantly increased PKC activity, which was reduced by EMPA and PKC inhibitor LY-333531. EMPA and LY-333531 showed a similar inhibitory capacity on NOX activity and ROS generation induced by 10 % stretch, which was not augmented by combined treatment with both drugs. PKC-β knockdown inhibits the NOX activation induced by Ca²⁺ and 10 % stretch. BAPTA, pharmacologic or genetic NCX inhibition and cariporide reduced Ca²⁺ in static HCAECs and prevented the activation of PKC and NOX in 10%-stretched cells. Ouabain increased ROS generation in cells exposed to 5 % stretch.

Conclusion: EMPA reduced NOX activity via attenuation of the NHE/Na⁺/NCX/Ca²⁺/PKC axis, leading to less ROS generation in HCAECs exposed to 10 % stretch.

Keywords: Cyclic stretch; Human coronary artery endothelial cells (HCAECs); Intracellular calcium (Ca(2+)); Nicotinamide adenine dinucleotide phosphate oxidase (NOX); Reactive oxygen species (ROS); Sodium glucose co-transporter 2 inhibitor (SGLT2i).