Abstract
Background: Diabetes is one of the risk for macrovascular complications. Dipeptidyl peptidase-4 (DPP-4) inhibitors are generally used for patients with type 2 diabetes. Previous reports revealed that DPP-4 inhibitors suppressed the progression of atherosclerosis in several atherosclerotic mouse models. However, the exact mechanisms for DPP-4 inhibitor-mediated suppression of atherosclerotic lesion formation have not been clarified yet. Therefore, to investigate the mechanism of anti-atherosclerotic effect of DPP-4 inhibitors, we studied the effect of linagliptin, one of the DPP-4 inhibitors, on atherosclerosis lesion formation in high fat diet (HFD)-fed apolipoprotein-E-deficient (ApoE KO) mice, and explored polarization of  atheromatous tissue-isolated macrophages (AIM) and bone marrow-derived macrophages(BMM) with linagliptin.
Methods: At 12 weeks of age, HFD-fed ApoE KO mice were treated with linagliptin (10 mg/kg/day) for 8 weeks. Body weight, amount of food intake and casual blood glucose levels were measured at least once a week. After the treatment, the mice were given intraperitoneal glucose tolerance tests (injected glucose: 1.5 g/kg) and intraperitoneal insulin tolerance tests (injected insulin: 1 U/kg) and were sacrificed to collect their aortas. The whole aortas and aortic sinuses were stained with Oil red O. We also collected serum from the mice, and measured serum lipid profiles by gel filtration HPLC (Skylight Biotech Inc, Akita, Japan). Whole aortas were used to evaluate expressions of mRNA for iNOS, MCP-1, TNF-α as M1 macrophage markers, and for arginase-1, Mgl2 and Ym1 as M2 macrophage markers by real-time RT-PCR. Macrophage polarization of AIMs in aortas were examined by FACS scan using anti-iNOS antibody for M1 macrophages and anti-CD206 antibody for M2 macrophages. Mouse BMMs were used in in vitro study. Expressions of iNOS, MCP-1, TNF-α, arginase-1, Mgl2, Ym1 mRNA and macrophage polarization were also evaluated by real-time RT-PCR and by FACS scan.
Results: There were no significant differences in food intake, body weight, blood glucose levels between each group, but cholesterol concentrations in VLDL and LDL particle were lower in the linagliptin group than those of the control group. Oil-Red-O staining demonstrated reduced atherosclerotic lesions size in the linagliptin group. M2 macrophage marker, arginase-1, Mgl2 and Ym1 mRNA expression in the aorta were higher in linagliptin groups than those in the control groups. However, M1 macrophage marker, MCP-1, TNF-α and iNOS mRNA expression were comparable. FACS scan revealed that the ratio of M2 macrophages in AIM was increased in linagliptin group than in control group, however, the ratio of M1 macrophages was not changed. In in vitro assay using BMMs, treatment with LPS and IFNγ increased MCP-1, TNF-α and iNOS mRNA expression, and linagliptin had no effect on the expression of these M1 markers. On the other hand, treatment with LPS and IFNγ decreased arginase-1, Mgl2 and Ym1 mRNA expression, and linagliptin significantly recovered these reductions. Treatment with LPS and IFNγ also decreased the ratio of M2 macrophages in AIM, and linagliptin ameliorated these reductions.
Conclusion: We revealed that linagliptin suppressed the progression of atherosclerosis in HFD-fed apoE KO mice. Moreover, linagliptin suppressed the decrease in M2 macrophage polarization. These findings may indicate the beneficial effects of DPP-4 inhibitors on the progression of diabetic macrovascular complications.

Inhibition of DPP-4 suppresses atherosclerotic lesion formation through the regulation of M2 macrophage polarization