Sodium-Glucose Cotransporter 2 Inhibitors regulate vascular cell function
Authors:   , Behnammanesh Ghazaleh 
Published: September 2024

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Vascular disease is the major cause of morbidity and mortality in diabetic patients. Although numerous factors contribute to the development of diabetes-associated vascular disease, the malfunction of vascular smooth cells (SMCs) and endothelial cells (ECs) plays a fundamental role. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are the latest class of glucose-lowering agents. By blocking glucose uptake in the proximal tubules of the nephron they induce glycosuria leading to decreases in circulating glucose. Notably, recent long-term, large scale cardiovascular outcome trials with SGLT2 inhibitors demonstrated benefits beyond glycemic control with a significant reduction in cardiovascular disease and mortality in type 2 diabetic patients. However, among the SGLT2 inhibitors, canagliflozin caused a 2-fold increase in limb amputations that was not seen with other SGLT2 inhibitors. The decrease in mortality by SGLT2 inhibitors has been attributed to improvements in renal function, energy balance, body weight, and blood pressure, but direct effects by these drugs on vascular cells have not been considered. In the present study, we investigated the effect of three US Food and Drug Administration approved SGLT2 inhibitors on cultured vascular cell function. Treatment of human umbilical vein ECs with canagliflozin (0-50[mu]M) resulted in a potent concentration-dependent inhibition in cell proliferation and DNA synthesis. The anti-proliferative effect by canagliflozin was observed at clinically attainable concentrations (5-10[mu]M) and this was not seen with empagliflozin or dapagliflozin. In addition, pharmacologically relevant concentrations of canagliflozin inhibited the proliferation of human and mouse aortic ECs. In contrast, only high concentration of canagliflozin or empagliflozin (30-50[mu]M) inhibited the migration of ECs. Flow cytometry experiments revealed that canagliflozin arrested ECs in the G0/G1 phase of the cell cycle, and this was paralleled by a decrease in the fraction of cells in S phase. Canagliflozin strongly inhibited the expression of cyclin A protein and the phosphorylation of retinoblastoma protein but minimally affected the expression of cyclin D1, cyclin E, and the cyclin-dependent kinase inhibitors p27 and p21. Infection of ECs with an adenovirus expressing cyclin A restored cyclin A protein expression in the presence of canagliflozin and partially rescued EC growth in the canagliflozin-treated cells. In addition, canagliflozin blocked EC tube formation in a concentration-dependent manner starting at a clinically relevant concentration of 10 [mu]M. Canagliflozin also inhibited the sprouting of ECs from rat aortic rings embedded in matrigel. Finally, incubation of rat aortic SMCs with a pharmacological concentration of canagliflozin (10 [mu]M), a near-pharmacological concentration of empagliflozin (5 [mu]M) and a supra-pharmacological concentration of dapagliflozin (50 [mu]M) inhibited cell proliferation, but only canagliflozin retarded vascular SMC migration. Analysis of SMC cell cycle distribution indicated that canagliflozin blocked cell cycle progression, as reflected by a decrease in the percentage of cells in S phase. In conclusion, pharmacologically relevant concentrations of canagliflozin inhibits human EC proliferation, in part, by suppressing cyclin A expression and prevents EC differentiation into tubes. The ability of canagliflozin to repress EC replication and tube formation may contribute to the increased risk of limb amputation in diabetic patients by impairing angiogenesis. Moreover, the ability of canagliflozin and perhaps empagliflozin to inhibit the proliferation of vascular SMCs may promote the beneficial cardiovascular actions of these drugs.