E could not clarify the mechanism by which linagliptin inhibited the AGE-induced ROS generation in HUVECs. However, we have previously shown that GLP-1 and GIP protect against AGE-induced HUVEC damage via anti-oxidative properties through the elevation of cyclic AMP, whose effect is augmented by the addition of DPP-4 inhibitor [27,38,39]. Since AGE-RAGE axis evokes ROS generationIshibashi et al. Cardiovascular Diabetology 2013, 12:125 http://www.cardiab.com/content/12/1/Page 7 ofFigure 4 Possible crosstalk between AGE-RAGE axis and soluble DPP-4.in ECs via NADPH oxidase activity, which is blocked by cAMP-elevating agents [6,7,15,40], linagliptin could enhance the beneficial effects of incretins on AGE-exposed HUVECs by inhibiting NADPH oxidase activity. Furthermore, we have recently found that linagliptin contains xanthine scaffold structure, which could inhibit xanthine oxidase activity in vitro [41]. The anti-oxidative unique properties of this drug might also be involved in the blockade of vicious cycle between ROS generation and RAGE gene induction. It is unlikely that linagliptin directly inhibited the AGE-RAGE interaction because highly sensitive 27-MHz quartz crystal microbalance analysis (Affinix Q; Initium, Tokyo, Japan) revealed that PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28607003 linagliptin can not bind to AGEs in vitro (data not shown). One early phase of atherosclerosis involves the recruitment and firm adhesion of inflammatory cells to ECs, whose process is mediated by adhesion molecules such as ICAM-1 [42,43]. Further, attenuated fibrinolytic activity due to increased PAI-1 levels is prevalent in diabetic patients, thus contributing to the increased risk of atherothrombosis in these subjects [37,44,45]. Linagliptin may be a promising strategy for not only ameliorating hyperglycemia in type 2 diabetic patients, but also protecting against vascular injury by suppressing ICAM-1 and PAI-1 expression through blockade of the deleterious effects of AGE-RAGE axis partly via inhibition of DPP-4 and M6P/IGF-IIR interaction. In pre-specified meta-analysis of cardiovascular events in linagliptin or comparator-treated patients with type 2 diabetes mellitus, the order Leupeptin (hemisulfate) hazard ratio for a composite of cardiovascular death, stroke, myocardial infarction, and hospitalization for unstable angina showed significantly lower risk with linagliptin than comparator [46]. Moreover, we have very recently found that DPP-4 inhibitor alogliptintreatment blocks the AGE-RAGE axis and resultantly reduces albuminuria in type 2 diabetes patients [47]. Fluorescent AGE levels have also been shown to be an independent marker of post-infarction heart failure development risk [48]. These data reinforce the important clinical implications of the present findings of linagliptin. The peak plasma concentration of linagliptin after administration of single oral dose of 5 mg is reported to be about 10 nM [49]. So, the concentration of linagliptin having beneficial effects on HUVECs used in the present experiments (10 nM) may also be comparable to the therapeutic level which is achieved in the treatment for patients with type 2 diabetes.LimitationsOur study has several limitations that should be noted. First, we did not examine here the effect of M6P/IGFIIR-Ab on the increase in ROS generation induced by AGEs or the increase in RAGE gene expression induced by DPP-4 and AGEs. Second, although mRNA levels of DPP-4 were not changed by the treatment with AGEs, the effect of linagliptin on membrane DPP-4 expression in AGE-ex.