Abstract
Angiopoietin-1 (Ang-1) is a ligand of Tie-2 receptors that promotes angiogenesis. It has been established that regulatory loops exist between angiogenic growth factors and distinct pro or anti-angiogenic miRNAs, but the nature and the roles of Ang-1-regulated miRNAs remain unclear. In this study, we assessed the role of miR-640 in Ang-1-induced angiogenesis in human umbilical vein endothelial cells (HUVECs). Exposure to Ang-1 (300 ng/mL) from 6 to 72 h significantly decreased expression of mature miR-640, a response that was mediated by Tie-2 receptors and was also observed in response to Ang-2, the vascular endothelial growth factor, and transforming growth factor β. Increasing miR-640 levels using a mimic inhibited Ang-1-induced cell migration and capillary-like tube formation whereas inhibition of miR-640 enhanced these responses. Pull down assays of biotinylated miR-640 revealed that miR-640 directly targets Zinc Finger Protein 91 (ZFP91), an atypical E3-ubiquitin ligase. Ang-1 exposure induced ZFP91 expression through down-regulation of miR-640. Silencing of ZFP91 significantly inhibited Ang-1-induced cell migration and tube formation. We conclude that Ang-1 upregulates ZFP91 expression through transcriptional down-regulation of miR-640 and that ZFP91 plays important roles in the promotion of Ang-1-induced endothelial cell migration and differentiation.
Highlights
Angiopoietin-1 (Ang-1) and its receptor, Tie-2, are major regulators of physiological and pathological angiogenesis
MiR-640 inhibited pro-migration andbasal pro-differentiation effectsand of Ang-1 but had no effects to PBS (Figure siRNA, cell migration capillary-like tube formation on cell survival; (3) zinc finger protein 91 (ZFP91) was a direct target of miR-640; (4) Ang-1 upregulated ZFP91 expression measured with PBS were significantly lower than those measured with scrambled siRNA (Figure 7C,D)
By decreasing miR-640 binding to ZFP91 mRNA; and (5) ZFP91 was required for Ang-1-induced cell migration and capillary-like tube formation
Summary
Angiopoietin-1 (Ang-1) and its receptor, Tie-2, are major regulators of physiological and pathological angiogenesis. In cultured endothelial cells (ECs), Ang-1 inhibits inflammation and apoptosis and stimulates differentiation, sprouting, and migration [2]. Ang-1 stimulates vascular remodeling and wound healing, increases lymphangiogenesis [3,4,5], and inhibits apoptosis [6]. It promotes EC migration and differentiation, and attenuates permeability [7,8,9]. The Ang-1/Tie-2 axis stimulates the PI-3 kinase and three members of the mitogen activated protein kinases (MAPKs; Erk1/2, p38, and SAPK/JNK) pathways [2,10] and these pathways mediate the majority of angiogenic properties of this axis.
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