title: Ghrelin Promotes Functional Angiogenesis in a Mouse Model of Critical Limb Ischemia Through Activation of Proangiogenic MicroRNAs.
abstract: Current therapeutic strategies for the treatment of critical limb ischemia (CLI) have only limited success. Recent in vitro evidence in the literature, using cell lines, proposes that the peptide hormone ghrelin may have angiogenic properties. In this study, we aim to investigate if ghrelin could promote postischemic angiogenesis in a mouse model of CLI and, further, identify the mechanistic pathway(s) that underpin ghrelin's proangiogenic properties. CLI was induced in male CD1 mice by femoral artery ligation. Animals were then randomized to receive either vehicle or acylated ghrelin (150 μg/kg sc) for 14 consecutive days. Subsequently, synchrotron radiation microangiography was used to assess hindlimb perfusion. Subsequent tissue samples were collected for molecular and histological analysis. Ghrelin treatment markedly improved limb perfusion by promoting the generation of new capillaries and arterioles (internal diameter less than 50 μm) within the ischemic hindlimb that were both structurally and functionally normal; evident by robust endothelium-dependent vasodilatory responses to acetylcholine. Molecular analysis revealed that ghrelin's angiogenic properties were linked to activation of prosurvival Akt/vascular endothelial growth factor/Bcl-2 signaling cascade, thus reducing the apoptotic cell death and subsequent fibrosis. Further, ghrelin treatment activated proangiogenic (miR-126 and miR-132) and antifibrotic (miR-30a) microRNAs (miRs) while inhibiting antiangiogenic (miR-92a and miR-206) miRs. Importantly, in vitro knockdown of key proangiogenic miRs (miR-126 and miR-132) inhibited the angiogenic potential of ghrelin. These results therefore suggest that clinical use of ghrelin for the early treatment of CLI may be a promising and potent inducer of reparative vascularization through modulation of key molecular factors.
authors.affiliation: Department of Physiology, HeartOtago (R.K., S.R., P.E.M., P.D., D.O.S.), University of Otago, Dunedin, 9010 New Zealand; Department of Cardiac Physiology (H.T., T.I., Y.F., M.S.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan; Japan Synchrotron Radiation Research Institute (K.U.), Hyogo, 679-5198 Japan; and Director (K.K.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Katare
authors.affiliation: Department of Physiology, HeartOtago (R.K., S.R., P.E.M., P.D., D.O.S.), University of Otago, Dunedin, 9010 New Zealand; Department of Cardiac Physiology (H.T., T.I., Y.F., M.S.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan; Japan Synchrotron Radiation Research Institute (K.U.), Hyogo, 679-5198 Japan; and Director (K.K.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan.
authors.forename: Shruti
authors.initials: S
authors.lastname: Rawal
authors.affiliation: Department of Physiology, HeartOtago (R.K., S.R., P.E.M., P.D., D.O.S.), University of Otago, Dunedin, 9010 New Zealand; Department of Cardiac Physiology (H.T., T.I., Y.F., M.S.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan; Japan Synchrotron Radiation Research Institute (K.U.), Hyogo, 679-5198 Japan; and Director (K.K.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan.
authors.forename: Pujika Emani
authors.initials: PE
authors.lastname: Munasinghe
authors.affiliation: Department of Physiology, HeartOtago (R.K., S.R., P.E.M., P.D., D.O.S.), University of Otago, Dunedin, 9010 New Zealand; Department of Cardiac Physiology (H.T., T.I., Y.F., M.S.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan; Japan Synchrotron Radiation Research Institute (K.U.), Hyogo, 679-5198 Japan; and Director (K.K.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan.
authors.forename: Hirotsugu
authors.initials: H
authors.lastname: Tsuchimochi
authors.affiliation: Department of Physiology, HeartOtago (R.K., S.R., P.E.M., P.D., D.O.S.), University of Otago, Dunedin, 9010 New Zealand; Department of Cardiac Physiology (H.T., T.I., Y.F., M.S.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan; Japan Synchrotron Radiation Research Institute (K.U.), Hyogo, 679-5198 Japan; and Director (K.K.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan.
authors.forename: Tadakatsu
authors.initials: T
authors.lastname: Inagaki
authors.affiliation: Department of Physiology, HeartOtago (R.K., S.R., P.E.M., P.D., D.O.S.), University of Otago, Dunedin, 9010 New Zealand; Department of Cardiac Physiology (H.T., T.I., Y.F., M.S.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan; Japan Synchrotron Radiation Research Institute (K.U.), Hyogo, 679-5198 Japan; and Director (K.K.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Fujii
authors.affiliation: Department of Physiology, HeartOtago (R.K., S.R., P.E.M., P.D., D.O.S.), University of Otago, Dunedin, 9010 New Zealand; Department of Cardiac Physiology (H.T., T.I., Y.F., M.S.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan; Japan Synchrotron Radiation Research Institute (K.U.), Hyogo, 679-5198 Japan; and Director (K.K.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan.
authors.forename: Parul
authors.initials: P
authors.lastname: Dixit
authors.affiliation: Department of Physiology, HeartOtago (R.K., S.R., P.E.M., P.D., D.O.S.), University of Otago, Dunedin, 9010 New Zealand; Department of Cardiac Physiology (H.T., T.I., Y.F., M.S.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan; Japan Synchrotron Radiation Research Institute (K.U.), Hyogo, 679-5198 Japan; and Director (K.K.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan.
authors.forename: Keiji
authors.initials: K
authors.lastname: Umetani
authors.affiliation: Department of Physiology, HeartOtago (R.K., S.R., P.E.M., P.D., D.O.S.), University of Otago, Dunedin, 9010 New Zealand; Department of Cardiac Physiology (H.T., T.I., Y.F., M.S.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan; Japan Synchrotron Radiation Research Institute (K.U.), Hyogo, 679-5198 Japan; and Director (K.K.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Kangawa
authors.affiliation: Department of Physiology, HeartOtago (R.K., S.R., P.E.M., P.D., D.O.S.), University of Otago, Dunedin, 9010 New Zealand; Department of Cardiac Physiology (H.T., T.I., Y.F., M.S.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan; Japan Synchrotron Radiation Research Institute (K.U.), Hyogo, 679-5198 Japan; and Director (K.K.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan.
authors.forename: Mikiyasu
authors.initials: M
authors.lastname: Shirai
authors.affiliation: Department of Physiology, HeartOtago (R.K., S.R., P.E.M., P.D., D.O.S.), University of Otago, Dunedin, 9010 New Zealand; Department of Cardiac Physiology (H.T., T.I., Y.F., M.S.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan; Japan Synchrotron Radiation Research Institute (K.U.), Hyogo, 679-5198 Japan; and Director (K.K.), National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan.
authors.forename: Daryl O
authors.initials: DO
authors.lastname: Schwenke
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D054439:Ghrelin / Q000008:administration & dosage / Q000494:pharmacology / Q000627:therapeutic use*; D006614:Hindlimb / Q000098:blood supply* / Q000473:pathology; D006801:Humans; D007511:Ischemia / Q000188:drug therapy*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000098:blood supply / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D018919:Neovascularization, Physiologic / Q000187:drug effects* / Q000235:genetics; D012039:Regional Blood Flow / Q000187:drug effects* / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D015533:Transcriptional Activation / Q000187:drug effects; D014664:Vasodilation / Q000187:drug effects
chemical_list: D054439:Ghrelin; D035683:MicroRNAs