S100A4 gene silencing in oxygen-induced ischemic retinopathy inhibits retinal neovascularization via down-regulation of CREB expression.

Purpose: To investigate the possible role of S100A4 in retinal neovascularization (RNV), as well as the underlying mechanism, in a mouse model of oxygen-induced retinopathy (OIR).

Methods: Retinas used in the experiments were obtained from a mouse model of OIR with and without treatment by intravitreal injection of adnoviral-S100A4-RNAi or adenoviral-green fluorescent protein at postnatal day 12 (P12). At P17, the efficacy of adenoviral gene transfer was assessed with fluorescence microscopy. RNV was evaluated by whole-mount immunofluorescent staining of the mouse retina with Griffonia simplicifolia isolectin B4 conjugated to Alexa Fluor 594 and quantified by counting the number of pre-retinal neovascular cells. Protein and mRNA expression levels of S100A4, cyclic AMP (cAMP) response element-binding protein (CREB), B-cell lymphoma 2 (Bcl-2), and caspase-3 were determined using Western blot analysis and real-time PCR.

Results: Retinal S100A4 levels were positively correlated with the progression of RNV. In the intravitreal Ad-S100A4-RNAi transfer mice, both S100A4 protein and mRNA expression levels in the retina were significantly decreased at P17. Ad-S100A4-RNAi transfer was clearly demonstrated by the green fluorescent protein (GFP) in many layers of the retina, including the ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), and outer nuclear layer (ONL), 3 days after intravitreal injection. Whole-mount immunofluorescent staining of the retina and quantification of the pre-retinal neovascular cells demonstrated that RNV was significantly ameliorated. At the same time, the expression of CREB and Bcl-2 were significantly decreased at the transcriptional and translational levels in the OIR-S100A4 group, whereas caspase-3 expression was increased.

Conclusions: Our results indicated that RNV was ameliorated by Ad-S100A4-RNAi transfer in a mouse model of OIR through mediation of the anti-apoptotic effect of Bcl-2 by reducing the expression of CREB, and that S100A4 may be a novel therapeutic target for ocular neovascularization diseases.