Comparative analysis of Retinal Angiogenesis during Inflammatory stage

Choroidal neovascularization (CNV) and the resulting retinal angiogenesis are pathological hallmarks of wet Age-related macular degeneration (AMD). The pathogenesis of CNV is not fully understood, but accumulated evidence has suggested the role of inflammation in the early stage of CNV. To better understand the molecular landscape during the early stage, we performed RNA-Seq and mass spectrometry-based proteomic analysis in the retina of the laser-induced CNV mouse model. Both transcriptomic and proteomic data showed dramatic activation of inflammatory response 3 days post photocoagulation. Integrative analysis suggested a moderate correlation between RNA-Seq and mass spec. Up-regulation of angiogenic factor, basic fibroblast growth factor-2 (Fgf-2), but not vascular endothelial growth factor (Vegf) was observed at both RNA and protein levels, highlighting Fgf-2 as a biomarker and potential therapeutic target during the early stage of CNV. In addition, enrichment analysis indicated a large overlap of inflammation-related genes and pathways at both levels. We also compared our findings with human retinal RNA-Seq data from AMD patients and controls. By using a multi-omics and comparative approach, our findings demonstrate the molecular landscape during the inflammatory stage of mouse CNV and provided new insight into the translation from the mouse model to understanding human AMD and its potential intervention and therapies. C57BL/6 mice (8-12 weeks) were pursued from Zhaoyan New Drug Research Center. The animals were maintained (5 mice/cage) in an air-conditioned room (24 ± 1°C) with a 12-h light/12-h dark cycle and free access to food and water and were allowed to acclimatize for 1 week before the experiment began. All animal experiments were performed by the requirements of the Animal Welfare Committee of Wuxi School of Medicine, Jiangnan University. All mice were anesthetized by intraperitoneal injection of hydrated chloral. Then pupils were dilated with topical administration of compound tomidlamide eye drops. Laser photocoagulation (75 um spot size, 0.1s duration) was conducted by 532nm laser (Quantel medical, VITRA, France) with slit lamp microscope (Haag-Streit, BQ900, Germany) at the 3, 6, 9, and 12 o’clock positions to burn the retina. For each mouse, only the right eye (oculus dextrus, OD) was undertaken laser induction. The left eye (oculus sinister, OS) was used as an autologous negative control. Three days and seven days post laser induction, mice were sacrificed and retinas were isolated for RNAseq.