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

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-ß 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.

Canagliflozin inhibits inflammasome activation in diabetic endothelial cells - Revealing a novel calcium-dependent anti-inflammatory effect of canagliflozin on human diabetic endothelial cells.

BACKGROUND: Canagliflozin (CANA) shows anti-inflammatory and anti-oxidative effects on endothelial cells (ECs). In diabetes mellitus (DM), excessive reactive oxygen species (ROS) generation, increased intracellular calcium (Ca2+) and enhanced extracellular signal regulated kinase (ERK) 1/2 phosphorylation are crucial precursors for inflammasome activation. We hypothesized that: (1) CANA prevents the TNF-α triggered ROS generation in ECs from diabetic donors and in turn suppresses the inflammasome activation; and (2) the anti-inflammatory effect of CANA is mediated via intracellular Ca2+ and ERK1/2. METHODS: Human coronary artery endothelial cells from donors with DM (D-HCAECs) were pre-incubated with either CANA or vehicle for 2 h before exposure to 50 ng/ml TNF-α for 2-48 h. NAC was applied to scavenge ROS, BAPTA-AM to chelate intracellular Ca2+, and PD 98059 to inhibit the activation of ERK1/2. Live cell imaging was performed at 6 h to measure ROS and intracellular Ca2+. At 48 h, ELISA and infra-red western blot were applied to detect IL-1ß, NLRP3, pro-caspase-1 and ASC. RESULTS: 10 µM CANA significantly reduced TNF-α related ROS generation, IL-1ß production and NLRP3 expression (P all <0.05), but NAC did not alter the inflammasome activation (P > 0.05). CANA and BAPTA both prevented intracellular Ca2+ increase in cells exposed to TNF-α (P both <0.05). Moreover, BAPTA and PD 98059 significantly reduced the TNF-α triggered IL-1ß production as well as NLRP3 and pro-caspase-1 expression (P all <0.05). CONCLUSION: CANA suppresses inflammasome activation by inhibition of (1) intracellular Ca2+ and (2) ERK1/2 phosphorylation, but not by ROS reduction.