Stimulation of endothelial progenitor cells by microRNA-31a-5p to induce endothelialization in an aneurysm neck after coil embolization by modulating the Axin1-mediated β-catenin/vascular endothelial growth factor pathway

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OBJECTIVE

Emerging evidence shows that frequent recurrence of intracranial aneurysms (IAs) after endovascular coiling is attributable to the lack of endothelialization across the aneurysm neck. Recently, much attention has been given to the role of microRNAs (miRs) in vascular disease, although their contributory role to IA is poorly understood.

METHODS

Adult male Sprague-Dawley rats were subjected to microsurgery to create a coiled embolization aneurysm model, and were injected with miR-31a-5p agomir or a negative control agomir via the tail vein at a dose of 10 mg/kg per week for 4 weeks after IA induction. H & E staining, scanning electron microscopy, and flow cytometry were performed to evaluate the effects of miR-31a-5p agomir on endothelialization and the number of circulating endothelial progenitor cells (EPCs). The effects of miR-31a-5p on the viability and functioning of EPCs were also determined using Cell Counting Kit–8, wound-healing assay, and tube formation assays.

RESULTS

The authors tested the ability of miR-31a-5p to promote EPC-induced endothelialization in a model of coiled embolization aneurysm. miR-31a-5p agomir improved endothelialization and elevated the number of circulating EPCs in the peripheral blood compared to a negative control agomir–treated group. In addition, the number of vWF- and KDR-positive cells in the aneurysm neck was increased in the miR-31a-5p agomir–treated group. Furthermore, upregulation of miR-31a-5p promoted EPC proliferation, migration, and tube formation and enhanced the expression of the proangiogenic factor vascular endothelial growth factor in vitro. Mechanistically, miR-31a-5p directly targeted the 3′ untranslated region (3′UTR) of Axin1 messenger RNA and repressed its expression. Besides, miR-31a-5p exerted its effect on EPCs by regulating the Axin1-mediated Wnt/β-catenin pathway.

CONCLUSIONS

Collectively, these results indicate that miR-31a-5p is an important regulator of EPC mobilization and endothelialization and may have a positive effect on aneurysm repair.

ABBREVIATIONS AN = aneurysm model; EPC = endothelial progenitor cell; IA = intracranial aneurysm; miR = microRNA; mRNA = messenger RNA; mt = mutant; NC = negative control; qRT-PCR = quantitative reverse transcription polymerase chain reaction; SEM = scanning electron microscopy; siRNA = signaling RNA; VEGF = vascular endothelial growth factor; wt = wild type; 3′UTR = 3′ untranslated region.

Article Information

Correspondence Wei Zhu: Huashan Hospital of Fudan University, Shanghai, People’s Republic of China. drzhuwei@fudan.edu.cn.

INCLUDE WHEN CITING Published online August 9, 2019; DOI: 10.3171/2019.5.JNS182901.

G.Y. and P.L. contributed equally to this work.

Disclosures The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Effects of miR-31a-5p on the endothelialization of the coil-treated aneurysm neck. A: Creation of the coiled embolization aneurysm model and a photomicrograph of an aneurysm neck section stained with H & E (original magnification ×10). B: The aneurysm neck sections from the AN/miR-31a-5p agomir and AN/NC agomir groups were examined after H & E staining, and the aneurysm repair scores were determined in these 2 groups of rats. C: Endothelialization was measured in the aneurysm neck by SEM analysis. D: Photomicrographs showing the vWF+ and KDR+ cells covering the bottom of the aneurysm. Cells are stained with DAPI (blue) and for vWF (green) and KDR (red). E and F: Quantification for the vWF+ and KDR+ cells per high-power field (HPF) in the AN/miR-31a-5p agomir group and AN/NC agomir group. *p < 0.05 versus the AN/NC agomir group. Figure is available in color online only.

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    The circulating EPC count was determined. A: The number of circulating EPCs was determined on days 10, 20, and 30 in groups designated as normal, AN/NC agomir, and AN/miR-31a-5p agomir. B: Quantification of CD34+KDR+ cells in these 3 groups. *p < 0.05 versus the normal group. #p < 0.05 versus the AN/NC agomir group. Figure is available in color online only.

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    miR-31a-5p promoted EPC proliferation, migration, and tube formation. A: Fluorescence photomicrographs show the results of FITC-UEA-I and DiI Ac-LDL assays and a merged image from confocal microscopy. B: Flow cytometry analysis of the expression of cell surface markers in EPCs. Cells were positive for both CD34 and KDR. C: miR-31a-5p expression was measured in EPCs transfected with miR-31a-5p mimics or an NC. D–F: Cell proliferation, migration, and tube formation were assessed using CCK-8, wound-healing, and tube formation assays, respectively. G: Relative tube length is shown. H: The VEGF levels in the culture supernatants from cultivated EPCs were detected on day 3. *p < 0.05 versus the control group. OD = optical density. Figure is available in color online only.

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    miR-31a-5p–activated Wnt/β-catenin signaling through repressing Axin1 expression. A: The alignment of putative binding sites for miR-31a-5p in Axin1 3′UTRs. B: A luciferase reporter assay was performed on HEK293T cells cotransfected with miR-31a-5p mimics and wt- or mt-Axin1 3′UTR. C: Axin1 mRNA expression was measured by qRT-PCR. D: Axin1 and nuclear β-catenin expression were detected in the EPCs transfected with miR-31a-5p by Western blotting with β-actin and lamin as a loading control. E: Expression levels of TCF4, c-Myc, and cyclin D1 were measured in EPCs by Western blotting. F and G: Immunohistochemical staining for Axin1 and β-catenin in the lumen of the aneurysm sac of groups AN/NC agomir and AN/miR-31a-5p agomir. *p < 0.05 versus the control group. Figure is available in color online only.

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    Axin1 abrogates the effects of miR-31a-5p on cell proliferation, migration, and tube formation. A: Axin1 and nuclear β-catenin expression was detected in the EPCs (transfected with the miR-31a-5p inhibitor or both miR-31a-5p inhibitor and Axin1 siRNA) by Western blotting with β-actin and lamin as a loading control. B–D: Cell proliferation, migration, and tube formation were detected. E: VEGF levels were determined by an enzyme-linked immunosorbent assay. F: The expression of TCF4, c-Myc, and cyclin D1 was measured by Western blotting. *p < 0.05 versus the NC group. #p < 0.05 versus the miR-31a-5p inhibitor group. Figure is available in color online only.

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