Analysis of protein expression profiling in a mouse model of oxygen-induced retinopathy

Retinal neovascularization (RNV) is one of the major causes of blindness worldwide, but the pathogenic mechanism of this disease remains unclear, and therapeutic modalities need to be improved.Therefore, it is necessary to identify ocular protein markers with significant expression changes during RNV, thereby providing novel therapeutic targets for neovascular retinopathies.

This study was to investigate retinal vessel morphological characteristics and protein expression profiling in a mouse model of oxygen-induced retinopathy (OIR).

Forty four C57BL/6J mouse pups were randomly divided into normal control group and OIR group at postnatal day 7 (P7). The mice in the normal control group were raised under the normal air for 10 days.The mice of the OIR group were exposed to (75±2)% oxygen for 5 days.The mother mice were alternated between the two groups every day.The mice of the OIR group returned to normal air at P12.Fluorescein isothiocyanate-dextran (FITC-dextran) was retrobulbarly injected at P17 mice from both groups, and the retinal flatmounts were prepared after fixation.The FITC-dextran-labeled retinal vessels were observed and quantified; the paraffin sections of eyeballs were processed for hematoxylin and eosin staining, and the number of pre-retinal vascular cell nuclei was quantified.The total proteins were extracted from the eyes, and the expression profiling was analyzed by a customized protein array and verified by Western blot and ELISA.

The retinal flatmounts labeled with FITC-dextran showed that the peripheral retinal microvessels in the OIR group were tortuous, disorganized with neovascular buds, and the vascular obliteration was prominent in the center of retina.Contrastly, the vessels were smooth, organized, and evenly distributed in the normal control group.The percentage of vascular obliteration area in the OIR group was (25.53±2.16)%, which was significantly higher than (0.66±0.36)% in the normal control group (t=-27.61, P<0.01). The number of pre-retinal vascular cell nuclei, as revealed by hematoxylin and eosin staining, was (28.41±3.97)/slide in the OIR group, which was substantially higher than (0.16±0.31)/slide in the normal control group (t=-54.42, P<0.001). Protein array showed that 10 out of the 62 examined pro-inflammatory, pro-angiogenic cytokines exhibited more than 1.5-fold expression changes, including 3 up-regulated cytokines and 7 down-regulated cytokines; 4 cytokines showed more than 2-fold expression changes, in which 3 cytokines were down-regulated and 1 cytokine was up-regulated.The differential expressions were verified by Western blot and ELISA.The expression trends of platelet factor 4 (PF-4), vascular endothelial growth factor A (VEGF-A), selectin P (SELP), vascular cell adhesion molecule 1 (VCAM-1), soluble tumor necrosis factor receptor Ⅱ (sTNF-RⅡ) and chemokine C-X-C motif ligand 16 (CXCL16) were consistent with those revealed by protein array.PF-4, VEGF-A and SELP were up-regulated, and the other 3 were significantly down-regulated (all at P<0.05).

Differential expression patterns of the cytokines, including PF-4, VEGF-A, SELP, VCAM-1, sTNF-RⅡ and CXCL16, are identified between normal and OIR mouse eyes.These differential expression patterns suggest that under the condition of OIR, the platelet system is activated, and proinflammatory factors are down-regulated.PF-4 might become a new target for VEGF-independent therapeutic strategy against RNV.