Figure 6.

Impaired lesion regression in diabetic mice is due to increased monocyte recruitment.

(A) Ldlr^−/− mice were fed a HCD for 16wks to develop atherosclerotic lesions. Aortic transplantation model overview: All donor Ldlr^−/− mice were injected with EdU (1 mg, i.v) to label newly formed monocytes. 48 hrs post-injection, aortas were dissected from these mice and either processed for baseline measurement of EdU⁺ cells or transplanted into chow fed Akita mice ± SGLT2i. 48hrs prior to termination, mice were injected with green fluorescent microspheres to determine monocyte entry.

(B) Monocyte entry determined by fluorescent beads.

(C-D) Monocyte egress: (C) Representative images showing EdU stain (red) in the aortic arch sections of donor mice (baseline) and grafts from Akita and Akita+SGLT2i.

(D) Quantification of monocyte egress as represented by the number of EdU⁺ cells per section. *P<0.05 vs. baseline, n=5/group.

(E) Diet induced regression model overview: Ldlr^−/− mice fed with a HCD for 16 wks were divided into 3 groups; regression (reg), reg+STZ and reg+STZ+SGLT2i and placed on chow diet. At the end of 4 weeks, all mice were injected (i.v) with clodronate liposomes (CLO, 250μl) to deplete the circulating monocytes. 48 hrs later they were injected with green fluorescent microspheres. 4 days later, a portion for mice from each group were sacrificed to determine the baseline measurement of beads in the atheroma and to assess Ly6-C^hi monocyte entry. A second group of mice was assessed 14 days later (6 weeks of regression) for quantification of labeled macrophages remaining in plaques. 48 hrs prior to sacrificing the final group of mice they were injected with EdU to also assess Ly6-C^hi monocyte entry.

(F) Ly6-C^hi monocyte entry as determined by EdU⁺ cells in the lesion.

(G) Ly6-C^hi monocyte entry and monocyte/macrophage retention in the lesion as assessed by number of beads/section.

(H) Percentage of monocyte/macrophage egress compared to baseline. *P<0.05 vs. all groups, ^P<0.05 final vs. baseline, n=6/group.

All values are means ± SEM.