The Mechanism Of Mitochondrial Fission Induced By Caffeine In Angiogenesis

BackgroundIschemic diseases are a group of disorders caused by the obstruction or stenosis of arterioles and capillaries that could not be compensated for by collateral circulation or vessel dilatation,which are a leading cause of disability and death around the world.Angiogenesis is a crucial process in new blood vessel formation and the subsequent expansion of vascular networks,and is triggered by ischemic conditions.Thus,angiogenesis is important for physiological recovery and functional protection of ischemic tissues.Mitochondria are the primary energy-generating organelles in most eukaryotic cells and have been widely reported to play a pivotal role in modulating angiogenesis via regulating the migration,proliferation and survival of endothelial cells,which form the inner layer of blood vessels.Mitochondria exist in the form of dynamic networks that change shape and intracellular distribution frequently,and the dynamics is precisely controlled by two opposite processes:fusion and fission.Previous studies have reported that increased mitochondrial fission plays an important role in breast cancer cell migration and invasion as well as vascular smooth muscle cell migration.However,whether alterations in mitochondrial dynamics and energy metabolism contribute to angiogenesis is unknown.Caffeine,as a constitute of coffee,tea,carbonated beverages,and a wide variety of medications,induces several physiological and pharmacological effects.However,till now little is known about its role in angiogenesis.Previous studies have demonstrated that caffeine increases mitochondrial function in Alzheimer’s mice and cells,and improves the functional capacity of endothelial cells in a mitochondria-dependent process,suggesting that caffeine may regulate mitochondrial function.Nevertheless,whether caffeine plays a critical role in angiogenesis via modulating mitochondrial dynamics is not understood.ObjectivesThis study aims to investigate the effect of caffeine on the behaviors of endothelial cell in the period of angiogenesis and the mechanisms of mitochondrial dynamics and bioenergetics.MethodsEndothelial cell proliferation,migration and tube formation were evaluated to determine the association between caffeine and angiogenesis.ELISA,western blot,agonists and inhibitors of signal molecular and RNA interference were used to determine the role of c AMP / PKA / AMPK / MFF signaling pathway in caffeine-induced angiogenesis.Mitochondrial energetics was assayed using a Seahorse Extracellular Flux Analyzer.8-week-old C57BL/6 male mice were used to establish a femoral artery ligation model of hind-limb ischemia to evaluate the relationship between caffeine and blood flow recovery after tissue ischemia.Immunofluorescence staining was used to detect the morphology of mitochondria,lamellipodia formation of endothelial cells,the density of capillaries and arterioles in the ischemic hind-limb gastrocnemius muscle.ResultsCaffeine was able to significantly facilitate angiogenesis in vitro,which was evidenced by concentration-dependent increases in tube formation of HUVECs and cell migration,but had no effects on cell proliferation.Caffeine at the concentration of 50 μM promoted endothelial migration through activating the c AMP/PKA/AMPK signaling pathway.This effect could be mimicked by c AMP analog 8-Br-c AMP,and blunted by PKA inhibitor H89,adenylate cyclase inhibitor SQ22536 or AMPK inhibitor compound C.Additionally,caffeine at the concentration of 50 μM induced prominent mitochondrial fragmentation via increasing the phosphorylation of mitochondrial fission protein dynamin-related protein 1(Drp1)in HUVECs,which improved its activity to modulate mitochondrial fission.The pharmacological blockade of Drp1 by Mdivi-1(10 μM)or the interference of mitochondrial fission by Drp1 silencing significantly inhibited caffeine-induced lamellipodia formation as well as endothelial cell migration.In addition,we demonstrated that caffeine-induced mitochondrial fission could lead to mitochondria accumulation in lamellipodia regions and augmentation of mitochondrial energetics,both of which were essential for endothelial cell migration.In the mouse hindlimb ischemia model,administration of caffeine significantly promoted angiogenesis and perfusion and the activation of vascular endothelial AMPK signaling in the ischemic hindlimbs.In summary,caffeine exerted the induction of mitochondrial fission via c AMP/PKA/AMPK/MFF signaling pathway.Mitochondrial fission was involved in the integral process during caffeine-induced endothelial cell migration by altering mitochondrial distribution and bioenergetics.Conclusions1.Caffeine at low concentrations induced mitochondrial fission in endothelial cells via c AMP/PKA/AMPK/MFF signaling pathway.2.Increased mitochondrial fission enhanced mitochondrial energetics and mediated mitochondrial redistribution in leading edge of endothelial cells to facilitate lamellipodia formation.3.Caffeine at low concentrations exerted angiogenic effect in endothelial cells in a mitochondrial fission-dependent manner.4.Caffeine treatment promoted angiogenesis and perfusion in ischemic hind-limb in a mouse model of hind-limb ischemia.