MicroRNA-223 Antagonizes Angiogenesis by Targeting β1 Integrin and Preventing Growth Factor Signaling in Endothelial Cells

Rationale: Endothelial cells in situ are largely quiescent, and their isolation and culture are associated with the switch to a proliferative phenotype. Objective: To identify antiangiogenic microRNAs expressed by native endothelial cells that are altered after isolation and culture, as well as the protein targets that regulate responses to growth factors. Methods and Results: Profiling studies revealed that miR-223 was highly expressed in freshly isolated human, murine, and porcine endothelial cells, but those levels decreased in culture. In primary cultures of endothelial cells, vascular endothelial cell growth factor and basic fibroblast growth factor further decreased miR-223 expression. The overexpression of precursor-miR-223 did not affect basal endothelial cell proliferation but abrogated vascular endothelial cell growth factor-induced and basic fibroblast growth factor-induced proliferation, as well as migration and sprouting. Inhibition of miR-223 in vivo using specific antagomirs potentiated postnatal retinal angiogenesis in wild-type mice, whereas recovery of perfusion after femoral artery ligation and endothelial sprouting from aortic rings from adult miR-223(-/y) animals were enhanced. MiR-223 overexpression had no effect on the growth factor-induced activation of ERK1/2 but inhibited the vascular endothelial cell growth factor-induced and basic fibroblast growth factor-induced phosphorylation of their receptors and activation of Akt. beta 1 integrin was identified as a target of miR-223 and its downregulation reproduced the defects in growth factor receptor phosphorylation and Akt signaling seen after miR-223 overexpression. Reintroduction of beta 1 integrin into miR-223-ovexpressing cells was sufficient to rescue growth factor signaling and angiogenesis. Conclusions: These results indicate that miR-223 is an antiangiogenic microRNA that prevents endothelial cell proliferation at least partly by targeting beta 1 integrin.