Sitagliptin Accelerates Endothelial Regeneration after Vascular Injury Independent from GLP1 Receptor Signaling

Abstract

Introduction: DPP4 inhibitors (gliptins) are commonly used antidiabetic drugs for the treatment of type 2 diabetes. Gliptins also act in a glucose-independent manner and show vasoregenerative effects. We have shown that gliptins can remarkably accelerate vascular healing after vascular injury. However, the underlying mechanisms remain unclear. Here, we examined potential signaling pathways linking gliptins to enhanced endothelial regeneration.

Methods and results: We used wild-type and GLP1 receptor knockout (Glp1r^-/-) mice to investigate the underlying mechanisms of gliptin-induced reendothelialization. The prototype DPP4 inhibitor sitagliptin accelerated endothelial healing in both animal models. Improved endothelial growth was associated with gliptin-mediated progenitor cell recruitment into the diseased vascular wall via the SDF1-CXCR4 axis independent of GLP1R-dependent signaling pathways. Furthermore, SDF1 showed direct proproliferative effects on endothelial cells. Excessive neointimal formation was not observed in gliptin- or placebo-treated Glp1r^-/- mice.

Conclusion: We identified the SDF1-CXCR4 axis as a crucial signaling pathway for endothelial regeneration after acute vascular injury. Furthermore, SDF1 can directly increase endothelial cell proliferation. Gliptin-mediated potentiation of endothelial regeneration was preserved in Glp1r^-/- animals. Thus, gliptin-mediated endothelial regeneration proceeds through SDF-1/CXCR4 in a GLP1R-independent manner after acute vascular injury.

Figure 1

Administration of oral sitagliptin over a period of three days significantly reduced serum DPP4-activity (^∗∗∗p ≤ 0.001).

Figure 2

(a) Sitagliptin treatment significantly improved endothelial regeneration compared to placebo treatment in both, Glp1r^−/− (GLP) and C57Bl/6N wild-type (Bl6) mice. Additional treatment with the CXCR4 receptor blocker AMD3100 abolished the positive gliptin effect completely (n.s. = not significant; ^∗∗p ≤ 0.01 and ^∗∗∗∗p ≤ 0.0001). (b) Evans blue staining of the injured carotid arteries three days after carotid injury (d0 = day 0; P = placebo; S = sitagliptin; AMD = AMD3100; and S & AMD = sitagliptin and AMD3100).

Figure 3

(a, b) The DPP4 inhibitor sitagliptin significantly enhanced the recruitment of CXCR4⁺CD133⁺ and CXCR4⁺Flk-1⁺ ciPC to the injured arterial walls. Additional administration of AMD3100 abolished the sitagliptin-mediated recruitment of ciPC (n.s. = not significant; ^∗p ≤ 0.05; and ^∗∗≤ 0.01). (c, d) The different treatments had no effect on the proportion of ciPC in the uninjured arterial walls (n.s. = not significant). (e) Representative dot plots from the FACS analyses.

Figure 4

(a–c) Weigert's Elastica van Gieson staining of the injured carotid arteries 28 days after vascular injury. (d, e) “Intima-to-media ratio” and “intima + media to total vessel cross section” were not affected by the different treatments (n.s. = not significant).

Figure 5

(a, b) Sitagliptin and sitagliptin + AMD3100 treatment had no significant influence on the total macrophage content in the injured and also in the uninjured carotid artery (n.s. = not significant). (c, d) The different treatments had no significant influence on the proportion of F4/80⁺Gr-1⁺ M1 and F4/80⁺CD206⁺ M2 macrophages (n.s. = not significant). (e) Representative dot plots from FACS analyses. Upper row shows F4/80⁺ total macrophages (right quadrant), and bottom row shows F4/80⁺Gr-1⁺ M1 (upper left quadrant) and F4/80⁺CD206⁺ M2 (bottom right quadrant) macrophages.

Figure 6

(a) 24 h incubation with 10 ng/ml and 100 ng/ml SDF1 leads to an increased scratch closure compared to 1 ng/ml SDF and placebo treatment (n.s. = not significant; ^∗∗∗∗p ≤ 0.0001). (b) Representative images of the scratch size immediately after scratch induction (left) and after 24 h (right).