Recent clinical trials revealed that sodium-glucose co-transporter 2 (SGLT2) inhibitors significantly reduce cardiovascular events in type 2 diabetic patients, however, canagliflozin increased limb amputations, an effect not seen with other SGLT2 inhibitors. Since endothelial cell (EC) dysfunction promotes diabetes-associated vascular disease and limb ischemia, we hypothesized that canagliflozin, but not other SGLT2 inhibitors, impairs EC proliferation, migration, and angiogenesis. Treatment of human umbilical vein ECs (HUVECs) with clinically relevant concentrations of canagliflozin, but not empagliflozin or dapagliflozin, inhibited cell proliferation. In particular, 10 μM canagliflozin reduced EC proliferation by approximately 45%. The inhibition of EC growth by canagliflozin occurred in the absence of cell death and was associated with diminished DNA synthesis, cell cycle arrest, and a striking decrease in cyclin A expression. Restoration of cyclin A expression via adenoviral-mediated gene transfer partially rescued the proliferative response of HUVECs treated with canagliflozin. A high concentration of canagliflozin (50 μM) modestly inhibited HUVEC migration by 20%, but markedly attenuated their tube formation by 65% and EC sprouting from mouse aortas by 80%. A moderate 20% reduction in HUVEC migration was also observed with a high concentration of empagliflozin (50 μM), while neither empagliflozin nor dapagliflozin affected tube formation by HUVECs. The present study identified canagliflozin as a robust inhibitor of human EC proliferation and tube formation. The anti-proliferative action of canagliflozin occurs in the absence of cell death and is due, in part, to the blockade of cyclin A expression. Notably, these actions are not seen with empagliflozin or dapagliflozin. The ability of canagliflozin to exert these pleiotropic effects on ECs may contribute to the clinical actions of this drug.

The use of HIV protease inhibitors (PIs) has extended the duration and quality of life for HIV-positive individuals. However there is increasing concern that this antiviral therapy may promote premature cardiovascular disease by impairing endothelial cell (EC) function. In the present study, we investigated the effect of HIV PIs on EC function and determined if the enzyme heme oxygenase (HO-1) influences the biological action of these drugs. We found that three distinct PIs, including ritonavir, atazanavir, and lopinavir, stimulated the expression of HO-1 protein and mRNA. The induction of HO-1 was associated with an increase in NF-E2-related factor-2 (Nrf2) activity and reactive oxygen species (ROS). PIs also stimulated HO-1 promoter activity and this was prevented by mutating the antioxidant responsive element or by overexpressing dominant-negative Nrf2. In addition, the PI-mediated induction of HO-1 was abolished by N-acetyl-L-cysteine and rotenone. Furthermore, PIs blocked EC proliferation and migration and stimulated the expression of intercellular adhesion molecule-1 and the adhesion of monocytes on ECs. Inhibition of HO-1 activity or expression potentiated the anti-proliferative and inflammatory actions of PIs which was reversed by bilirubin but not carbon monoxide. Alternatively, adenovirus-mediated overexpression of HO-1 attenuated the growth-inhibitory and inflammatory effect of PIs. In contrast, blocking HO-1 activity failed to modify the anti-migratory effect of the PIs. Thus, induction of HO-1 via the ROS–Nrf2 pathway in human ECs counteracts the anti-proliferative and inflammatory actions of PIs by generating bilirubin. Therapeutic approaches targeting HO-1 may provide a novel approach in preventing EC dysfunction and vascular disease in HIV-infected patients undergoing antiretroviral therapy.

Metformin is the drug of first choice in treating hyperglycemia in patients with type 2 diabetes mellitus. Its primary action is to inhibit hepatic glucose production and increase the sensitivity of peripheral tissues to insulin. Recently, sodium‐glucose cotransporter 2 inhibitors such as canagliflozin, which reduce blood glucose levels by blocking glucose reabsorption in the kidney, have also been approved for use in type 2 diabetes mellitus either alone or in combination with other drugs, including metformin. Intriguingly, both metformin and canagliflozin have been shown to improve cardiovascular outcomes in patients with type 2 diabetes mellitus. While reductions in body weight, adiposity, blood pressure, and arterial stiffness have been suggested to contribute to the cardiovascular benefit of these drugs, their effects on vascular cell function have not been fully considered. In the present study, we tested the hypothesis that metformin and canagliflozin directly effect human endothelial cell function. Treatment of human umbilical vein endothelial cells with metformin or canagliflozin inhibited their proliferation in a concentration‐dependent manner. The anti‐proliferative action of both drugs was associated with a significant reduction in DNA synthesis and the arrest of endothelial cells in the G0/G1 phase of the cell cycle. While pharmacologically relevant concentrations of canagliflozin (10μM) blocked endothelial cell proliferation, much higher supra‐pharmacological concentrations of metformin were needed to repress endothelial cell growth. In addition, both drugs inhibited the migration of endothelial cells and the differentiation of endothelial cells into tubes in a concentration‐dependent fashion. Furthermore, incubation of endothelial cells with a clinically attainable concentration of metformin (10μM) potentiated the ability of canagliflozin to block endothelial cell proliferation, but not endothelial cell migration or differentiation. In conclusion, metformin and canagliflozin inhibits human endothelial cell proliferation, migration, and tube formation. Moreover, metformin potentiates the anti‐proliferative action of canagliflozin, demonstrating a novel and clinically relevant interaction between these two anti‐diabetic agents on endothelial cell function. The ability of these drugs to modify endothelial cell function either individually, or in combination, may contribute to their cardiovascular actions in patients with type 2 diabetes mellitus.Support or Funding InformationAmerican Diabetes Association Grant #1‐17‐IBS‐290.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.