Effects Of Ang/Tie 2 And VEGF/VEGFR Signals On Retinal Neovascularization And Vascular Development

Part 1:An appropriate mouse model of retinal neovascularization and vascular developmentBackgroup and purpose:The basic pathological changes of oxygen-induced retinopathy (OIR) animal model is that immature retina presents ischemic and hypoxic, which leads to retinal neovascularization (RNV) under high-oxygen environment. This model also demonstrates retinal vascular development, which is similar to retinopathy of prematurity (ROP). And the Smith method has good feasibility and repeatability. So we choose this OIR mouse model to build RNV and retinal vascular development.Methods:7 day old C57BL/6J mice were fed for 5 days in a high-oxygen container with a concentration of 75%±2% and moved to normal air environment, which were defined as OIR group. Age-related C57BL/6J mouse were raised to 17 day old in the normal environment, which were defined as control group. Two groups of mice were performed cardiac FITC-dextran perfusion and retinal flat-mount. Then total retinal area, nonperfusion area, micro vessel density of perfusion area, neovascularization cluster, venous expansion, and arterial tortuosity were compared between two groups. Endothelial cells were counted and compared.Results:Compared with the control group, the OIR group presented decreased total retinal area ((54.5 ± 5.2× 105 pixel values vs.60.7 ± 1.5 ×105 pixel values, P=0.0004), bigger nonperfusion area (25.5±5.4% vs.0 ± 0%, P<0.0001), higher microvessel density of perfusion area (30.9 ± 1.7% vs.48.6 ± 4.9%, P<0.0001), more neovascularization cluster (0±0% vs.2.6±1.3%,P<0.0001), obvious expansion of vein (9.2±0.9 pixel values vs.17.0±1.5 pixel values, P<0.0001), and tortuosity of arteries (1.02±0.01vs.1.14±0.04,P<0.0001). The OIR group also demonstrated more endothelial cells than the control group (49.6±6.8 vs.0.6±1.1, P<0.0001).Conclusions:The OIR mouse model was an appropriate model of RNV and vascular development and this model was repetitive and could be analyzed quantitatively.Part 2:Effects of Ang/Tie2 and VEGF/VEGFR signals on retinal neovascularization and vascular developmentBackgroup and purpose:The most important vascular growth factors include vascular endothelial growth factor (VEGF) and angiopoietin (Ang). Soluble Tie2 fusion protein (sTie2-Fc) and Angl could antagonize the neovascularization effect of Ang2. Ranibizumab is a kind of VEGF antibodies, which could bind to all kind of VEGFA without Fc. So phosphate buffer saline (PBS), sTie2-Fc, Angl, Ranibizumab, sTie2-Fc+Ranibizumab or Angl+Ranibizumab was intravitreal injected into one eye of mice of every group and the different influence on RNV and vascular development were compared among different groups.Methods:82 OIR mice were divided into 6 groups (14,14,13,14,14,13). At 12 days old, all mice were moved out of high-oxygen container and intravitreal injected one kind of reagents 1μl in one eye (PBS, sTie2-Fc 0.05μg, Angl 0.05μg, Ranibizumab 5μg, sTie2-Fc 0.05μg+Ranibizumab 5μg or Angl 0.05μg+Ranibizumab 5μ.g). Then weigh of every mouse was measured and documented. All mice were raised in the air environment till 17 days old. After measuring of weight, all mice were sacrificed. Cardiac FITC-dextran perfusion and retinal flat-mount were performed to measure total retinal area, nonperfusion area, microvessel density of perfusion area, neovascularization cluster. And endothelial cells were counted. The results were compared among different groups.Results:1. Compared with the PBS group, sTie2-Fc group, Angl group, Ranibizumab goup, sTie2-Fc+Ranibizumab group, and Angl+Ranibizumab group all demonstrated decreased neovascularization area (P value was<0.0001, <0.0001,<0.0001, 0.0001, and<0.0001 respectively) and less endothelial cells(P value was<0.0001, 0.0001,0.0001,0.0001, and 0.0001 respectively). What was more, sTie2-Fc+Ranibizumab group and Angl+Ranibizumab group showed greater effect on the inhibition of neovascularization.2. Compared with PBS group, sTie2-Fc group, Angl group, Ranibizumab goup, and Angl+Ranibizumab group presented less nonperfusion area (P value was 0.0001,0.003,0.01, and 0.01 respectively) and decreased microvessel density (P value was 0.0001,0.0001,0.0001, and 0.0001 respectively). But sTie2-Fc+Ranibizumab group had an even increased nonperfusion area and less microvessel density then other five groups.3. Only Ranibizumab group showed less total retinal area (P value was 0.0001,0.02,0.0001,0.003, and 0.0001 respectively) and weight at 17 days old than other groups (P value was 0.0001,0.008. 0.001,0.01, and 0.02 respectively).Conclusions:Intravitreal injection of sTie2-Fc, Angl, or Ranibizumab could inhibit RNV without influence of retinal vascular development. Among them, sTie2-Fc could even promote vascular development. Combined intravitreal injection of sTie2-Fc+ Ranibizumab or Angl+Ranibizumab presented greater inhibition of RNV, but the former one cold also inhibited vascular development, although the later did not, which might be a better choice of combination treatment.