Glucose lowering by SGLT2-inhibitor empagliflozin accelerates atherosclerosis regression in hyperglycemic STZ-diabetic mice

Abstract

Diabetes worsens atherosclerosis progression and leads to a defect in repair of arteries after cholesterol reduction, a process termed regression. Empagliflozin reduces blood glucose levels via inhibition of the sodium glucose cotransporter 2 (SGLT-2) in the kidney and has been shown to lead to a marked reduction in cardiovascular events in humans. To determine whether glucose lowering by empagliflozin accelerates atherosclerosis regression in a mouse model, male C57BL/6J mice were treated intraperitoneally with LDLR- and SRB1- antisense oligonucleotides and fed a high cholesterol diet for 16 weeks to induce severe hypercholesterolemia and atherosclerosis progression. At week 14 all mice were rendered diabetic by streptozotocin (STZ) injections. At week 16 a baseline group was sacrificed and displayed substantial atherosclerosis of the aortic root. In the remaining mice, plasma cholesterol was lowered by switching to chow diet and treatment with LDLR sense oligonucleotides to induce atherosclerosis regression. These mice then received either empagliflozin or vehicle for three weeks. Atherosclerotic plaques in the empagliflozin treated mice were significantly smaller, showed decreased lipid and CD68⁺ macrophage content, as well as greater collagen content. Proliferation of plaque resident macrophages and leukocyte adhesion to the vascular wall were significantly decreased in empagliflozin-treated mice. In summary, plasma glucose lowering by empagliflozin improves plaque regression in diabetic mice.

Figure 1

Applying antisense/sense oligonucleotides and the SGLT2 inhibitor empagliflozin to regulate plasma cholesterol and glucose levels. (A) Timeline of atherosclerosis regression study. Wildtype mice received weekly ip. injections of LDLR-/SRBI- antisense and HCD during the atherosclerosis progression period and were subjected to five consecutive STZ-injections at week 14. An atherosclerosis baseline group was harvested in week 16. Atherosclerosis regression was then initiated by LDLR sense treatment and switching to chow diet. All mice received either the SGLT2 inhibitor empagliflozin or vehicle. (B) Total plasma cholesterol during atherosclerosis progression and regression, inlets on the right show plasma levels at 16 weeks and 19 weeks. (C) Total plasma triglyceride levels during atherosclerosis progression and regression. (D) Body weight and (E) 4-hour fasting plasma glucose after STZ-treatment (n = 8–11/group). ns = not significant. Error bars represent SEM.

Figure 2

Diminished atherosclerotic plaque size in SGLT2 inhibitor-treated mice. Mean of aortic root lesion area, n = 7–9/group. *p < 0.05, ***p < 0.001. Error bars represent SEM.

Figure 3

SGLT2-inhibitor treatment accelerates features of plaque stability during atherosclerosis regression. (A) Quantification of lipid content of aortic root plaques by ORO-staining, 4 sections/mouse, n = 7–9. (B) Lipid content at 0 µm, 32 µm, 72 µm, 112 µm. (C) Representative pictures of ORO-stained aortic root sections. (D) Quantification of CD68⁺ macrophages in aortic root sections. 1 section/mouse, n = 7–9/group. (E) Representative pictures of respective aortic root sections and magnifications, n = 7–9/group. *p < 0.05, ***p < 0.001. Error bars represent SEM. (F) Quantification of collagen content of aortic root plaques by Picrosirius-Red staining, 2 sections/mouse, n = 7–9. (G) Representative pictures of Picrosirius-Red-stained aortic root sections. **p < 0.01. ns = not significant. Error bars represent SEM.

Figure 4

Leukocytes and leukocytes subsets are not affected by the SGLT2-inhibitor empagliflozin during atherosclerosis regression. (A) Quantification by flow cytometry of total circulating leukocytes, (B) leukocytes subsets, (C) patrolling, “non-classical” Ly6C^lo and (D) pro-inflammatory Ly6C^hi monocytes. (E) regulatory FoxP3⁺ T lymphocytes. N = 7–9/group. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars represent SEM.

Figure 5

Glucose lowering by the SGLT2-inhibitor empagliflozin alleviates proliferation of plaque resident macrophages. (A) Quantification of proliferating Ki67⁺ macrophages in atherosclerotic lesions. N = 7–9/group (B) Representative immunofluorescence staining of the aortic root of baseline, control and SGLT2i mice. Proliferating macrophages were stained with anti-CD68 (red), anti-Ki67 (green), and 4’,6-diamidino-2-phenylindole (DAPI; blue) monoclonal antibodies. White dashed line represents border of the atherosclerotic plaque to the vessel lumen. Red scale = 20 µm (C) Correlation between plasma glucose and proliferating, plaque-resident macrophages in the two regression groups. *p < 0.05, **p < 0.01. ns = not significant. Error bars represent SEM.

Figure 6

SGLT2 inhibition leads to decreased leukocyte adhesion in vivo. (A) Timeline of the intravital microscopy (IVM) study. Male BL/6J WT-mice were rendered diabetic by 5 consecutive STZ injections at age 6 weeks. 10 days later 4-hour-fasting-plasma glucose was measured. Mice with glucose levels >250 mg/dl were included into the study. Mice then received either the SGLT2i empagliflozin or control. After one week IVM was performed. (B) 4-hour fasting plasma glucose levels ten days after STZ injection (before SGLT2i Tx) and seven days after initiation of SGLTi treatment. (C) Quantification of leukocyte adhesion in intestinal venules assessed by intravital microscopy. (D) Quantification of rolling leukocytes in intestinal venules by intravital microscopy, (n = 8–10/group). (E) Representative still images of videos acquired by intravital microscopy (40x). (F) Quantification of blood leukocytes before and after i.p. injection of TNFα for IVM (n = 11–13/group). *p < 0.05, ***p < 0.001. ns = not significant. Error bars represent SEM.