Purpose:Hyperglycemia (HG)-induced oxidative stress and inflammation are major determinants contributing to vascular cell dysfunction and subsequent cardiovascular events in Type II Diabetes mellitus (T2DM). The selective sodium-glucose co-transporter-2 (SGLT-2) inhibitor, Empagliflozin, is known to improve cardiovascular mortality in T2DM patients (EMPA-REG trial). Since SGLT-2 is expressed on monocytes and endothelial cells (EC), we investigated the potential ability of empagliflozin to regulate glucose transport and alleviate HG-induced dysfunction of these cells.

Methods:Primary human monocytes were isolated from T2DM patients and healthy individuals. Primary human umbilical vein endothelial cells (HUVECs) and primary human coronary artery endothelial cells (HCAECs) were used as EC model cells. Cells were exposed to HG conditions in vitro in the presence of 40 or 100 ng/ml empagliflozin. The expression levels of SGLT-2 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 via FACS and fluorescence microscopy. Monocyte and endothelial cell chemotaxis were measured using modified Boyden chamber assays. To quantify the expression of other relevant molecules, RT-qPCR and FACS were performed.

Results:Both, primary human monocytes and endothelial cells express SGLT-2. A difference in transcripts of SGLT-2 in T2DM monocytes could not be detected. In vitro, HG conditions did not significantly alter SGLT-2 levels in both monocytes and ECs. Glucose uptake assays carried out in the presence of GLUT inhibitors revealed that SGLT-2 inhibition very mildly suppresses glucose uptake (around 8-12%) but does not significantly alter the glucose uptake by monocytes or endothelial cells. However, we oberserved a significant suppression of HG-induced ROS accumulation in monocytes (1.3-fold, p=0.021) and ECs (1.5-fold, p=0.003) when Empagliflozin was present. HG monocytes and endothelial cells readily exhibited an impaired chemotaxis behaviour. The co-treatment with Empagliflozin reversed the PlGF-1 resistance phenotype of HG monocytes (1.4-fold, p=0.002). Similarly, the blunted VEGF-A responses of HG ECs were restored by Empagliflozin (1.35-fold, p=0.028), which could be attributed to the restoration of the VEGFR-2 receptor levels on the EC surface. Induction of oxidative stress completely recapitulated most of the aberrant phenotypes exhibited by HG monocytes and endothelial cells. We could mimic these effects induced by Empagliflozin using the general antioxidant N-acetyl-L-cysteine (NAC).

Conclusions:This study provides data indicating the beneficial role of Empagliflozin in reversing HG-induced vascular cell dysfunction. Eventhough monocytes and endothelial cells express functional SGLT-2, it is not the primary glucose transporter in these cells. Therefore, it seems likely that HG-mediated enhanced glucotoxicity in these cells is not directly prevented by Empagliflozin through inhibition of glucose uptake. We identified the reduction of oxidative stress by Empagliflozin in HG conditions as a primary reason for the improved function of HG monocytes and endothelial cells. We conclude that independent of glucose transport, the antioxidant properties of Empagliflozin reverses vascular cell dysfunction. This property may also underlie the robust beneficial cardiovascular effects of this drug.