Pathological angiogenesis in the retina is one of the main ocular diseases closely associated with vision loss. This work investigated the roles of microRNA-34a (miR-34a) and its potential target Notch1, in retinal angiogenesis. For this we used oxygen-induced retinopathy (OIR) rats and human retinal microvascular endothelial cells (HRMECs) stimulated with vascular endothelial growth factor (VEGF). We performed hematoxylin–eosin staining, Western blot for VEGF, and immunofluorescence staining for CD31 to verify the establishment of our OIR model. We observed down-regulation of miR-34a, and up-regulation of Notch1 and Hey1 in retinas from OIR rats. We found similar results with the VEGF-stimulated HRMECs. By performing MTT assay, cell scratch assay, tube formation assay, and by detecting the expression of matrix-metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitors of metalloproteinases-1 (TIMP-1), and TIMP-2, we found that transfection of miR-34a ameliorated VEGF-mediated angiogenesis of HRMECs. We further observed that siRNA-induced gene silencing of Notch1 prevented VEGF-induced angiogenesis via regulating cell proliferation, cell migration, and tube formation of HRMECs. Additionally, activation of Notch1 by transfection of Notch1 plasmid attenuated the inhibitory effects of miR-34a on tube formation, in the present of VEGF. Results from our dual-luciferase reporter gene assay suggested that miR-34a targets Notch1. In summary, our data demonstrate that miR-34a attenuates retinal angiogenesis via targeting Notch1.

Abstract. Abnormal activation of Rho kinase (ROcK) plays a vital role in the pathogenesis of ischemia/reperfusion (I/R)-induced retinal injury. The aim of this study was to investigate whether fasudil, a potent inhibitor of ROcK, has a protective effect on retinal I/R injury in rats and to explore the possible underlying mechanisms. Forty adult Sprague-dawley rats were randomly assigned into sham, I/R injury model (I/R), model plus normal saline (control), and model plus fasudil (fasudil) groups. Rats in the control and fasudil groups were intravitreously injected with normal saline and fasudil, respectively, 5 min prior to the induction of ischemia. Retinal ischemia was induced by increasing the intraocular pressure to 100 mmHg for 60 min. Overall retinal thickness and retinal cell apoptosis was evaluated by histological analysis and the TUNEL assay, respectively. The protein expression of caspase-3 and the Bax/Bcl-2 mRNA ratio were also examined. Moreover, the retinal expression of inducible nitric oxide synthase (iNOS) was determined by immunohistochemical staining, quantitative real-time RT-PcR and Western blot analysis. Fasudil attenuated the I/R-induced apoptosis of retinal cells in the inner nuclear and ganglion cells of the rat retina. Fasudil significantly decreased the Bax/Bcl-2 mRNA ratio and the expression of caspase-3 and iNOS compared to the control group (P<0.05). Seven days after I/R, the overall retinal thickness in the fasudil group was significantly greater compared to that in the control group (P<0.05). In conclusion, fasudil can protect the rat retina from I/R injury by inhibiting apoptosis and iNOS expression, suggesting that fasudil may have a therapeutic potential for the prevention of retinal diseases associated with I/R.

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