Background: Ischemic retinopathies (IRs) are ocular disorders associated to microvascular degeneration leading to visual impairments and blindness. microRNA (miRNAs) are a family of non-coding RNAs that regulate a wide range of gene expression involved in various biological process such blood vessel development and pathological NV. However, the post-transcriptional modulation of miRs and especially, their specific functions in the eyes during IRs remain to be evaluated. We aim to evaluate the potential role of miR-96 on microvascular degeneration in a rat model of oxygen-induced retinopathy (OIR). Methods: In vivo: next generation sequencing (NSG) was used to perform a complete miRNAs profiling in the retina and choroid from OIR and normoxia (CTL) rats. To evaluate the effects of miR-96 on microvasculature, OIR animals were treated with a miR-96 mimic (1 mg/kg) or a control-miR by intravitreal injection before hyperoxia-exposure (80% O2). Immunostaining analysis of retinal flatmounts and cryosections was used to explore the microvascular effects of miR-96. In vitro: Human Retinal Microvascular Endothelial Cells (HRMVEC) were subjected or not to hyperoxia (80% O2) and transfected with 50 nM of miR-96 mimic or antagomir-96. Angiogenic assay was performed (tube formation and migration) and molecular analysis evaluated by qRT-PCR and western blot. Results: NSG and qRT-PCR analyses identified miR-96 as one of most highly expressed miRNAs in retina and choroid during development. However, miR-96 showed a strong downregulation in OIR rats, and also in HRMVEC subjected to hyperoxia. In HRMVEC, we found that miR-96 regulates positively the expression of the key pro-angiogenic factors VEGF, FGF-2 and ANG-2. To better explore the role of miR-96 on HRMVEC angiogenic activity, we performed a gain/loss of function study. Similarly, to hyperoxia exposure, we observed a robust angiogenic impairment (tube formation and migration) on HMRVEC transfected with an antagomiR-96. Interestingly, overexpression of miR-96 completely recued the basal phenotype of HRMVEC and protected against hyperoxia-induced endothelial dysfunction. In vivo, intravitreal injection of miR-96 mimic (1 mg/kg) in OIR rats significantly restored retinal vascular density and choroidal tightness/sprouting hability. This was accompanied by the restoration in the physiological levels of VEGF, FGF-2 and ANG-2. Conclusions: This is the first study showing that reduced expression of miR-96 in OIR conditions lead to a reduction of VEGF/FGF/ANG-2 signaling, and inneficient post-ischemic revascularization in retinal/choroidal tissues. Intravitreal supplementation of miR-96 using a miR mimic could constitute a novel therapeutic strategy to improve vascular repair in IRs.
Ischemic retinopathies (IRs) are leading causes of visual impairment. They are characterized by an initial phase of microvascular degeneration and a second phase of aberrant pre-retinal neovascularization (NV). microRNAs (miRNAs) regulate gene expression, and a number play a role in normal and pathological NV. But, post-transcriptional modulation of miRNAs in the eye during the development of IRs has not been systematically evaluated.Using Next Generation Sequencing (NGS) we profiled miRNA expression in the retina and choroid during vasodegenerative and NV phases of oxygen-induced retinopathy (OIR).Approximately 20% of total miRNAs exhibited altered expression (up- or down-regulation); 6% of miRNA were found highly expressed in retina and choroid of rats subjected to OIR. During OIR-induced vessel degeneration phase, miR-199a-3p, -199a-5p, -1b, -126a-3p displayed a robust decreased expression (> 85%) in the retina. While in the choroid, miR-152-3p, -142-3p, -148a-3p, -532-3p were upregulated (>200%) and miR-96-5p, -124-3p, -9a-3p, -190b-5p, -181a-1-3p, -9a-5p, -183-5p were downregulated (>70%) compared to controls. During peak pathological NV, miR-30a-5p, -30e-5p and 190b-5p were markedly reduced (>70%), and miR-30e-3p, miR-335, -30b-5p strongly augmented (by up to 300%) in the retina. Whereas in choroid, miR-let-7f-5p, miR-126a-5p and miR-101a-3p were downregulated by (>81%), and miR-125a-5p, let-7e-5p and let-7g-5p were upregulated by (>570%) during NV. Changes in miRNA observed using NGS were validated using qRT-PCR for the 24 most modulated miRNAs. In silico approach to predict miRNA target genes (using algorithms of miRSystem database) identified potential new target genes with pro-inflammatory, apoptotic and angiogenic properties.The present study is the first comprehensive description of retinal/choroidal miRNAs profiling in OIR (using NGS technology). Our results provide a valuable framework for the characterization and possible therapeutic potential of specific miRNAs involved in ocular IR-triggered inflammation, angiogenesis and degeneration.
