Figure 4.

Hyperglycaemia-induced monocyte dysfunction is reversed by empagliflozin through the modulation of oxidative stress. (A) CD14⁺⁺CD16⁻ monocytes were cultured in vitro in normoglycemic (5 mM glucose) or hyperglycaemic (30 mM glucose + 100 µM methylglyoxal) for 48 h and were analysed for their ability to undergo chemotaxis (directional migration) towards arteriogenic stimuli PlGF-1. Boyden chamber assays were performed. n = 6. (B) CD14⁺⁺CD16⁻ monocytes were cultured in vitro in normoglycemic (5 mM glucose) or hyperglycaemic (30 mM glucose + 100 µM methylglyoxal) for 48 h and were analysed for their ability to undergo chemokinesis (random migration). Checkerboard analyses were performed for this. n = 6. All data are means ± SEM. (C,D) CD14⁺⁺CD16⁻ monocytes were cultured in vitro in normoglycemic (5 mM glucose) conditions in the presence of 200 µM H₂O₂ for 24 h. After that, the cells were analysed for their ability to undergo chemotaxis towards PlGF-1 and chemokinesis using Boyden chamber assays. n = 6. All data are means ± SEM. (E) CD14⁺⁺CD16⁻ monocytes were cultured in vitro in normoglycemic (5 mM glucose) or hyperglycaemic (30 mM glucose + 100 µM methylglyoxal) for 48 h. The reactive oxygen species (ROS) accumulated was detected by fluorescence spectroscopy using 5-(and-6)-carboxy-2′,7′-difluorodihydrofluorescein diacetate (H2-DFFDA) reagent. n = 3. All data are means ± SEM. (F,G) CD14⁺⁺CD16⁻ monocytes were cultured in vitro under hyperglycaemic (30 mM glucose + 100 µM methylglyoxal) conditions for 48 h in the presence or absence of 100 ng/mL SGLT-2 inhibitor empagliflozin. After that, the cells were analysed for their ability to undergo chemotaxis towards PlGF-1 and chemokinesis using Boyden chamber assays. n = 6. All data are means ± SEM. ns = non-significant. * p < 0.05, ** p < 0.01 and *** p < 0.001.