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MiR-125a-5p impairs endothelial cell angiogenesis in aging mice via RTEF-1 downregulation.

ABSTRACT: Increasing evidence suggests that microRNAs (miRNAs) play important roles in impaired endothelial cell (EC) angiogenesis during aging. However, their exact roles in the aging process remain unclear. We aimed to determine whether miRNAs cause angiogenesis defects in ECs during aging and to uncover the underlying mechanisms. To study the miRNA-induced changes in ECs during aging, we performed microarray analyses on arterial ECs collected from young and aging mice. Using qRT-PCR, we showed that microRNA-125a-5p (mir-125a-5p) expression was approximately 2.9 times higher in old endothelial cells (OECs) compared with samples collected from young animals. Western blot assays showed a lower expression level of an mir-125a-5p target known as related transcriptional enhancer factor-1 (RTEF-1) in OECs compared with its expression levels in young cells. Overexpression of mir-125a-5p in young endothelial cells (YECs) using pre-mir-125a-5p caused the downregulation of RTEF-1, endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) and resulted in impaired angiogenesis, as evidenced by spheroid sprouting and tube formation assays in vitro. Conversely, repression of mir-125a-5p in OECs using anti-mir-125a-5p increased RTEF-1, eNOS and VEGF expression and improved EC angiogenesis. Importantly, impaired angiogenesis caused by knock-down of RTEF-1 was not efficiently rescued by anti-mir-125a-5p. Dual-luciferase reporter gene analysis showed that RTEF-1 is a direct target of mir-125a-5p, which regulates angiogenesis by repressing RTEF-1 expression and modulating eNOS and VEGF expression. These findings indicate that mir-125a-5p and RTEF-1 are potential therapeutic targets for improving EC-mediated angiogenesis in elderly individuals.

SUBMITTER: Che P

PROVIDER: S-EPMC4331751 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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miR-125a-5p impairs endothelial cell angiogenesis in aging mice via RTEF-1 downregulation.

Che Peng P Liu Jun J Shan Zhen Z Wu Ridong R Yao Chen C Cui Jin J Zhu Xiaonan X Wang Junwei J Burnett Mary Susan MS Wang Shenming S Wang JinSong J

Aging cell 20140724 5

Increasing evidence suggests that microRNAs (miRNAs) play important roles in impaired endothelial cell (EC) angiogenesis during aging. However, their exact roles in the aging process remain unclear. We aimed to determine whether miRNAs cause angiogenesis defects in ECs during aging and to uncover the underlying mechanisms. To study the miRNA-induced changes in ECs during aging, we performed microarray analyses on arterial ECs collected from young and aging mice. Using qRT-PCR, we showed that mic ...[more]

PMID: 25059272

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Project description:BackgroundAlthough neoangiogenesis is a hallmark of chronic inflammatory diseases such as inflammatory arthritis and many cancers, therapeutic agents targeting the vasculature remain elusive. Here we identified miR-125a as an important regulator of angiogenesis.MethodsMiRNA levels were quantified in Psoriatic Arthritis (PsA) synovial-tissue by RT-PCR and compared to macroscopic synovial vascularity. HMVEC were transfected with anti-miR-125a and angiogenic mechanisms quantified using tube formation assays, transwell invasion chambers, wound repair, RT-PCR and western blot. Real-time analysis of EC metabolism was assessed using the XF-24 Extracellular-Flux Analyzer. Synovial expression of metabolic markers was assessed by immunohistochemistry and immunofluorescent staining. MiR-125a CRISPR/Cas9-based knock-out zebrafish were generated and vascular development assessed. Finally, glycolytic blockade using 3PO, which inhibits Phosphofructokinase-fructose-2,6-bisphophatase 3 (PFKFB3), was assessed in miR-125a-/- ECs and zebrafish embryos.FindingsMiR-125a is significantly decreased in PsA synovium and inversely associated with macroscopic vascularity. In-vivo, CRISPR/cas9 miR-125a-/- zebrafish displayed a hyper-branching phenotype. In-vitro, miR-125a inhibition promoted EC tube formation, branching, migration and invasion, effects paralleled by a shift in their metabolic profile towards glycolysis. This metabolic shift was also observed in the PsA synovial vasculature where increased expression of glucose transporter 1 (GLUT1), PFKFB3 and Pyruvate kinase muscle isozyme M2 (PKM2) were demonstrated. Finally, blockade of PFKFB3 significantly inhibited anti-miR-125a-induced angiogenic mechanisms in-vitro, paralleled by normalisation of vascular development of CRISPR/cas9 miR-125a-/- zebrafish embryos.IntepretationOur results provide evidence that miR-125a deficiency enhances angiogenic processes through metabolic reprogramming of endothelial cells. FUND: Irish Research Council, Arthritis Ireland, EU Seventh Framework Programme (612218/3D-NET).

Project description:BackgroundVascular dementia (VaD) mainly results from cerebral vascular lesions and tissue changes, which contribute to neurodegenerative processes. Effective therapeutic approaches to targeting angiogenesis may reduce mortality of VaD. Endothelial progenitor cells (EPCs) play a key role in postnatal angiogenesis. Many exosomal microRNAs (exo-miRNAs) have been reported to involve in the development of dementia. The present study was designed to investigate whether the expression profile of the exo-miRNAs is significantly altered in patients with VaD and to reveal the function of differentially expressed miRNAs and the relevant mechanisms in EPC-mediated angiogenesis in VaD rat model.ResultsExosomes isolated from serum of patients with VaD (n = 7) and age-matched control subjects (n = 7), and miRNA sequencing and bioinformatics analysis found that circulating exosome miRNA-155-5p, miRNA-154-5p, miR-132-5p, and miR-1294 were upregulated in patients with VaD. The expression of miRNA-154-5p was further verified to be upregulated in clinical samples (n = 23) and 2-vessel occlusion-induced VaD rat model by reverse transcription quantitative PCR (RT-qPCR). Notably, miRNA-154-5p inhibition in bone marrow-EPCs (BM-EPCs) from VaD rats improved EPC functions, including tube formation, migration, and adhesion, and elevated concentrations of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α). The mRNA levels of ICAM-1, VCAM-1, and MCP-1 were reduced in miRNA-154-5p-inhibited EPCs. In addition, miRNA-154-5p inhibition increased the level of superoxide dismutase (SOD), and decreased reactive oxygen species (ROS) in EPCs. PRKAA2 was chosen as a promising target gene of miR-154-5p, and miRNA-154-5p inhibition upregulated the protein expression of AMPKα2. Furthermore, upregulation of miR-154-5p markedly diminished EPC functions and inhibited angiogenesis following EPC transplantation in VaD rats.ConclusionCirculating exo-miR-154-5p was upregulated in patients with VaD, and miR-154-5p upregulation was associated with impaired EPC functions and angiogenesis in VaD rat model. Therefore, miR-154-5p is a promising biomarker and therapeutic strategy for VaD.

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MiR-125a-5p impairs endothelial cell angiogenesis in aging mice via RTEF-1 downregulation.

Publications

miR-125a-5p impairs endothelial cell angiogenesis in aging mice via RTEF-1 downregulation.

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