Abstract 10854: Ghrelin Promotes Functional Angiogenesis in a Mouse Model Of Critical Limb Ischemia Through Activation of Pro-angiogenic MicroRNAs

Abstract

Background: 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. Objective: To investigate if ghrelin could promote post-ischemic angiogenesis in a mouse model of CLI and identify the mechanistic pathway(s) that underpin ghrelin’s pro-angiogenic properties. Methods and Results: CLI was induced in male CD1 mice by femoral artery ligation. Animals were then randomized to receive either vehicle or ghrelin (Ghr,150μg/kg/sc) for 14 consecutive days. Subsequently, Synchrotron Radiation microangiography was used to assess hindlimb perfusion. Ischemia alone stimulated the generation of new microvessels typically having an ID < 80 μm, however, the magnitude of ‘angiogenesis’ was significantly augmented in ghrelin-treated mice (13.9 ± 1.3 vessels in CLI+Ghr mice vs. 7.6 ± 0.9 vessels in CLI+Vehicle mice, P < 0.05). Moreover, the newly formed vessels of only the ghrelin-treated CLI mice were both structurally and functionally normal; evident by robust endothelium-dependent vasodilatory responses to acetylcholine. Histological analysis showed marked increase in the number of arterioles in Ghrelin treated group (81±8/mm 2 in vehicle treated group vs. 134±11/mm 2 in Ghrelin treated group, P<0.01, n=5), confirming the angiography data. Molecular analysis revealed that ghrelin’s angiogenic properties were linked to activation of pro-survival Akt/VEGF/Bcl-2 signaling cascade, thus reducing the apoptotic cell death and subsequent fibrosis. Furtherstill, ghrelin treatment activated pro-angiogenic (miR-126, -132 and -27a) and anti-fibrotic (miR-30a) microRNAs while inhibiting anti-angiogenic (miR-92a and -206) microRNAs. Further, in vitro knockdown of miR-126 and miR-132 in human umblical vein endothelial cells markedly attenuated the angiogenic potential of ghrelin, as evaluated by decreased proliferation and tube forming capacity (P<0.05). Conclusion: Ghrelin therapy significantly restored the blood flow to the ischemic hind limb by accelerating the formation of new functional arterioles through activation of pro-angiogenic microRNAs.