AMPK Inhibits ALK1-mediated Angiogenesis

Objectives: To explore negative regulation of ALK1-mediated signaling as well as the underlying mechanism and to evaluate the inhibitory effect of AMPK activators on ALK1-mediated angiogenesis.Background: Angiogenesis is an important physiological and pathological process.Dysregulated angiogenesis is implicated in cancer,diabetic eyes and age-related macular degeneration.Thus,inhibition of angiogenesis has emerged as an essential treatment strategy.Nowadays,anti-VEGF therapy has been applied for treatment of cancer and diabetic eyes.Although clinical practice shows effective in many cases,resistance or refraction develops eventually.Therefore,it is imperative to seek alternative therapeutic targets.ALK1 is a type I receptor for TGF-? /BMP superfamily,which has been shown to play an important role in angiogenesis induced by TGF-? family members.Recent studies have indicated that inhibition of ALK1-mediated signaling is an additional approach for treatment of tumor angiogenesis.Methods:(1)Western blot was used to examine the effect of AMPK activators on BMP-signaling in human umbilical endothelial cells(HUVEC);(2)Tube formation assay was conducted to assess the effect of AMPK activators on BMP9/ALK1-mediated angiogenesis;(3)Active mutant or dominant negative mutant of AMPK was delivered in adenovirus to HUVECs to assess their roles or responses to metformin in BMP9-induced tube formation;(4)active mutant of ALK1 was delivered in adenovirus to HUVECs to assess its response to metformin in tube formation;(5)Matrigel plug assay was conducted to assess the effect of metformin on BMP9-induced agiogenesis in vivo;(6)Laser-induced choroidal neovascularization was conducted to assess the effect of metformin and ALK1 inhibitor on neovascularization.Results: Treatment of HUVECs with metformin as well as other pharmacological AMPK activators inhibited Smad1/5 phosphorylation and tubeformation induced by BMP9 and active mutant of ALK1.This event was mimicked by expression of the active mutant of AMPK ? 1 and prevented by the dominant negative AMPK ? 1.Metformin inhibition of BMP9/ALK1 signaling is possibly mediated by upregulation of Smurf1,leading to degradation of ALK1.Furthermore,metformin suppressed BMP9-induced angiogenesis in mouse Matrigel plug.In addition,laser photocoagulation assay revealed that metformin significantly reduced choroidal neovascularization to a level comparable to LDN212854,an ALK1 specific inhibitor.In conjunction,metformin diminished expression of ALK1 in endothelium of the lesion area.Conclusions: Collectively,our study for the first time demonstrates that AMPK inhibits ALK1 and associated angiogenesis/neovascularization.This offers us a new avenue for the treatment of related diseases using metformin and other clinically used pharmacological AMPK activators in combination with other strategies to enhance the treatment efficacy or in the case of anti-VEGF resistance.