The Sodium-Glucose Co-Transporter 2 (SGLT2) Inhibitor Empagliflozin Reverses Hyperglycemia-Induced Monocyte and Endothelial Dysfunction Primarily through Glucose Transport-Independent but Redox-Dependent Mechanisms

Abstract

Purpose: Hyperglycaemia-induced oxidative stress and inflammation contribute to vascular cell dysfunction and subsequent cardiovascular events in T2DM. Selective sodium-glucose co-transporter-2 (SGLT-2) inhibitor empagliflozin significantly improves cardiovascular mortality in T2DM patients (EMPA-REG trial). Since SGLT-2 is known to be expressed on cells other than the kidney cells, we investigated the potential ability of empagliflozin to regulate glucose transport and alleviate hyperglycaemia-induced dysfunction of these cells. Methods: Primary human monocytes were isolated from the peripheral blood of T2DM patients and healthy individuals. Primary human umbilical vein endothelial cells (HUVECs) and primary human coronary artery endothelial cells (HCAECs), and fetoplacental endothelial cells (HPECs) were used as the EC model cells. Cells were exposed to hyperglycaemic conditions in vitro in 40 ng/mL or 100 ng/mL empagliflozin. The expression levels of the relevant molecules were analysed by RT-qPCR and confirmed by FACS. Glucose uptake assays were carried out with a fluorescent derivative of glucose, 2-NBDG. Reactive oxygen species (ROS) accumulation was measured using the H2DFFDA method. Monocyte and endothelial cell chemotaxis were measured using modified Boyden chamber assays. Results: Both primary human monocytes and endothelial cells express SGLT-2. Hyperglycaemic conditions did not significantly alter the SGLT-2 levels in monocytes and ECs in vitro or in T2DM conditions. Glucose uptake assays carried out in the presence of GLUT inhibitors revealed that SGLT-2 inhibition very mildly, but not significantly, suppressed glucose uptake by monocytes and endothelial cells. However, we detected the significant suppression of hyperglycaemia-induced ROS accumulation in monocytes and ECs when empagliflozin was used to inhibit SGLT-2 function. Hyperglycaemic monocytes and endothelial cells readily exhibited impaired chemotaxis behaviour. The co-treatment with empagliflozin reversed the PlGF-1 resistance phenotype of hyperglycaemic monocytes. Similarly, the blunted VEGF-A responses of hyperglycaemic ECs were also restored by empagliflozin, which could be attributed to the restoration of the VEGFR-2 receptor levels on the EC surface. The induction of oxidative stress completely recapitulated most of the aberrant phenotypes exhibited by hyperglycaemic monocytes and endothelial cells, and a general antioxidant N-acetyl-L-cysteine (NAC) was able to mimic the effects of empagliflozin. Conclusions: This study provides data indicating the beneficial role of empagliflozin in reversing hyperglycaemia-induced vascular cell dysfunction. Even though both monocytes and endothelial cells express functional SGLT-2, SGLT-2 is not the primary glucose transporter in these cells. Therefore, it seems likely that empagliflozin does not directly prevent hyperglycaemia-mediated enhanced glucotoxicity in these cells by inhibiting glucose uptake. We identified the reduction of oxidative stress by empagliflozin as a primary reason for the improved function of monocytes and endothelial cells in hyperglycaemic conditions. In conclusion, empagliflozin reverses vascular cell dysfunction independent of glucose transport but could partially contribute to its beneficial cardiovascular effects.

Transient inhibition of sodium-glucose cotransporter 2 after ischemia/reperfusion injury ameliorates chronic kidney disease

“…Finally, we also observed a reduction in renal inflammation with Dapa, as indicated by reduced myeloid infiltration, which was consistent with the reduction in mitochondrial NLRP ( 57 ). Although SGLT2 expression in leukocytes in vivo is unclear, there are studies suggesting a direct effect of flozins on monocytes and endothelial cells in models of ischemic myocardial injury and in hyperglycemic or septic in vitro assays ( 58 – 60 ). Interestingly, Dapa reduced TLR4 and NF-κB activation in differentiated macrophages and human endothelial cells stimulated with LPS ( 58 ).…”

“…In addition, this drug also suppressed M1 and promoted M2 macrophage polarization in postischemic hearts, suggesting a downregulation of inflammation ( 59 ). Moreover, incubation with empagliflozin reduced oxidative stress in human monocytes and endothelial cells exposed to hyperglycemic conditions, resulting in reduced chemotaxis and endothelial dysfunction ( 60 ). Although the reduced leukocyte infiltration observed in the IR + Dapa group may be an indirect effect of improved tubular repair, we cannot exclude a direct regulation of proinflammatory signals in immune and endothelial cells leading to an earlier resolution of inflammation with Dapa.…”

The Impact of Pharmacotherapy for Heart Failure on Oxidative Stress—Role of New Drugs, Flozins

“…Semo et al revealed that empagliflozin reversed hyperglycemia-induced monocyte and endothelial dysfunction despite the fact that SGLT2 is not the primary glucose transporter in these cells. Its antioxidant effect was similar to N-acetyl-L-cysteine [ 186 ]. One of the last experiments showed that empagliflozin suppressed mitochondrial reactive oxygen species generation and mitigated the inducibility of atrial fibrillation in diabetic rats [ 182 ].…”

Integrated approach to reducing polypharmacy in older people: exploring the role of oxidative stress and antioxidant potential therapy

“…Empagliflozin has also reported the decrease of superoxide and hydrogen peroxide induced by hyperglycemia in cultures of human proximal tubular epithelial cells, and in turn prevents the activation of inflammatory signaling pathways [ 304 ]. On the other hand, recent studies reported that empagliflozin would have antioxidant and anti-renal senescence effects mediated by SIRT1 in a d-galactose-induced aging model in rats [ 305 ] and also managed to restore endothelial dysfunction induced by hyperglycemia through the modulation of oxidative stress in human cell cultures [ 306 ].…”