The Role And Possible Mechanism Of Regulators Of G-protein Signaling 5 In The Formation Of Experimental Choroidal Neovascularization

Backgroud: Choroidal neovascularization (CNV) is one of the important manifestations of intraocular neovascularization and it may always be reason for visual impairment. The process of CNV formation influenced by many factors and cytokines is very complex and complicated and probably due to Bruch's membrane damage and hypoxia. The former can reduce the nutrient transport capacity and increase lipid deposition which leads to the rapture of Bruch's membrane, then gradually, proliferating choroidal vessels surmount Bruch's membrane and spread in the subpigment epithelial and subretinal space to form a CNV membrane; The latter can induce functional changes of retinal pigment epithelial (RPE) cells, releasing multiple cytokines, and destroy the integrity of RPE-Bruch membrane-choriocapillaris complex. Subsequently, endothelial cells may proliferate and migrate and new blood vessels will formate.Regulator of G-protein signalling 5 (RGS5) belongs to the R4/B subfamily of RGS proteins, and they are found to not only negatively regulate G protein signaling, but also inhibit the activation of mitogen activated protein kinases (MAPK) signal pathway. RGS5 connects with hypoxia and pathological neovascularization, and this effect may be related to vascular endothelial growth factor (VEGF). VEGF is secreted by RPE cells and one of the important factors in CNV formation. As an anti-VEGF recombinant monoclonal antibody, bevacizumab (Avastin) can block the binding of all VEGF-A isoforms and surface receptors of endothelial cells, and it can effectively prevent the formation of CNV by inhibiting VEGF activity.Objective: To observe the role and possible mechanism of the expression of RGS5 in experimental CNV formation and in human RPE cells under hypoxia comdition.Methods: (1) A total of 88 male Brown Norway (BN) rats underwent the 532nm photocoagulation to set up the CNV model. They were sort by time as follows: 1d (n=6), 3d (n=6), 7d (n=7) and 14d (n=21), then by treatment factor: intravitreal injection of physiological saline (n=6) and Avastin (n=6) as soon as induction of CNV for 7 days, and the same (n=18, n=18) for 14 days; (2) To set up the hypoxia model, human RPE cells were cultured with 200μM CoCl2. The RPE cells were sorted for showing the expression of RGS5 and VEGF and by time as follows: 0, 1, 3, 6, 12 and 24 hours, then by treatment factor: Hypoxia and VEGF inhibitor bevacizumab (Avastin) group and the concentration of Avastin included 25μg/ml, 100μg/ml and 250μg/ml. The expression of RGS5 and VEGF were examined by immunofluorescence, Western blot and RT-PCR. The thickness and the area of CNV were qualified by histopathological sections and choroidal flatmount. All the results were determined by Student's t-test.Results: (1) The expression of RGS5 protein and mRNA were decreased at 1day (t1d=3.451,P<0.05;t1d=3.646,P<0.05) and 3 days (t3d=3.085,P<0.05;t3d=3.888,P<0.05) after photocoagulation and reached a peak at 14 days (t14d=2.079,P<0.05;t14d=2.194,P>0.05); The expression of VEGF protein and mRNA reached a peak at 7 days (t7d=7.959,P<0.01;t7d=7.959,P<0.01) after photocoagulation; The thickness and the area of CNV were significantly decreased (t=2.616,P<0.05;t=15.179,P=0.000) after intravitreal injection with Avastin,and the expression of either the protein or the mRNA of VEGF and RGS5 was reduced; (2) RGS5 protein mainly distributed in the cytoplasm of cultured RPE cells under normoxic conditions. The expression of RGS5 protein and mRNA were increased under hypoxia conditions, and reached a peak in 24 hours after hypoxia (0.932±0.104, t₂₄=3.106, P=0.011; 0.742±0.083, t₂₄=2.852, P=0.017). The highest level of expression of VEGF protein and mRNA occurs in 12 hours (1.022±0.141, t₁₂=4.144, P=0.002; 0.491±0.063, t₁₂=5.707, P=0.000), and still higher than normal in 24 hours (0.942±0.125, t₂₄=3.306, P=0.008; 0.425±0.080, t₂₄=3.239, P=0.011). The expression of RGS5 protein and mRNA were inhibited by Avastin in the 100μg/ml (t₁₀₀=2.953, P=0.014; t₁₀₀=3.009, P=0.013) and 250μg/ml (t₂₅₀=2.913, P=0.015; t₂₅₀=4.243, P=0.002) group, so did the expression of VEGF (t₁₀₀=2.794, P=0.019; t₁₀₀=2.823, P=0.018; t₂₅₀=3.396, P=0.007; t₂₅₀=9.584, P=0.000).Conclusion: The expression of RGS5 and VEGF were related to the formation of CNV, and RGS5 expressed after VEGF. The expression of RGS5 was decreased when CNV was inhibited by anti-VEGF. On this basis, results in vitro suggest that the expression of VEGF increased before RGS5 under hypoxia condition, and Avastin can effectually inhibit the levels of RGS5 and VEGF expression in human RPE cells. To sum up, RGS5 may be located downstream of VEGF signaling pathway, so potentially, the expression of RGS5 may play a role in the mechanism of VEGF related CNV formation and orignate partially from RPE cells.