Metformin stimulates ischemia-induced revascularization through an eNOS dependent pathway in the ischemic hindlimb mice model

ObjectiveAs first-line treatment for type 2 diabetes, metformin has gained a strong position. In addition, type 2 diabetics benefit from the fact that metformin is associated with a reduction in cardiovascular events. Nevertheless, there is a dearth of information concerning the functional role of metformin in regulating angiogenesis. Our present study explores whether metformin is involved in the modulation of the revascularization processes in vivo by employing a hindlimb mice model of ischemia-induced angiogenesis.MethodsFor comparative purposes, randomly selected wild-type (WT) mice or endothelial nitric oxide synthase (eNOS) deficient mice were assigned to one of two groups. One group was orally administered a daily dose of metformin through a gastric tube whereas the other group served as a control with no metformin administered. Both groups were subjected to unilateral hindlimb ischemia. Laser Doppler analysis coupled with capillary density staining with CD31was the method employed to determine revascularization. Adenosine monophosphate-activated protein kinase (AMPK) and eNOS phosphorylation levels were assessed using Western blot analysis.ResultsSubsequent to hindlimb ischemic surgery, in comparison to the nontreated mice, metformin-treated WT mice showed accelerated limb perfusion, which was substantiated by laser Doppler blood-flow measurements and the presence of increased capillary density in the ischemic adductor muscle. Treatment with metformin significantly enhanced the increase in AMPK and eNOS phosphorylation levels of muscle tissues in WT mice induced by ischemia. In eNOS- deficient knockout mice, there was a significant increase in ischemic tissue AMPK phosphorylation induced by metformin; however, blood flow recovery in ischemic limb after surgery was unaffected.ConclusionsMetformin promoted revascularization in the presence of tissue ischemia through an AMPK/eNOS-related mechanism. Our study indicates that, in addition to its glucose-lowering effect, metformin fosters improved revascularization, which is responsible for its positive effect on patients with critical limb ischemia.

Clinical RelevanceIn patients with peripheral arterial disease, the incidence of the diabetes mellitus is high. One of the comorbidities that affect wound healing is diabetes. In Japan, in particular, the complication rates of diabetes mellitus and end-stage renal disease are high in patients with critical limb ischemia. Metformin is a widely used first-line drug for the treatment of type2 diabetes. The present study indicates that, in addition to its glucose-lowering effect, metformin has beneficial effects on the improvement of revascularization, which produce positive effects on the patient with critical limb ischemia.