| ?Objective?Retinal neovascularization (RNV) is a common pathological condition of ischemic retinopathy, as well as a leading cause of blindness in developed countries, and a main reason of visual loss in developing countries. Despite numerous studies have been performed, the molecular mechanisms underlying RNV, however, remain unclear. The cell adhesion kinase?(CAK?) is tyrosine phosphorylated and activated in response to elevation of [Ca2+]i or various stress signals. Functionally, CAK?is involved in cell adhesion and cytoskeletal organization via phosphorylation of paxillin. However, the effect of CAK?on RNV and its relationship to vascular endothelial growth factor (VEGF) is poorly understood. This study is designed to investigated whether RNV augmented the expression and phosphorylation of CAK?and VEGF, We also investigated the effects of Irbesartan and Fluvastatin on CAK?signalling pathway in RNV.?Methods?RNV was induced with the use of a mice model of oxygen-induced retinopathy (OIR) according to the protocol of Smith et al. (1) 80 P7 C57BL/6J mice were randomly assigned into 2 groups: control group (C group) and OIR group. Mice were euthanized at P12, P14, P17, and P19 respectively. (2) 180 P7 C57BL/6J mice were randomly assigned into 3 groups: C group, OIR group and BVZ group (intraocular injection of 5mg/Kg Bevacizumab at P12 in OIR mice). Mice were euthanized at P12, P14, and P17 respectively. (3) 140 P7 C57BL/6J mice were randomly assigned into 5 groups: C group, FVS group (intraocular injection of 10mg/Kg/d Fluvastatin from P12 to P17in OIR mice), IBS group (intraocular injection of 50mg/Kg/d Irbesartan from P12 to P17in OIR mice), FVS+ IBS group (intraocular injection of 10mg/Kg/d Fluvastatin+50mg/Kg/d Irbesartan from P12 to P17in OIR mice), and OIR group. Mice were euthanized at P17. (4) Retinal angiograohy and quantization were used to observe RNV. Intracellular reactive oxygen species (ROS) levels were measured using 2?,7?-dichlorofluorescein (DCF) diacetate probes. VEGF mRNA and CAK?mRNA expression were detected by Real-time RT-PCR, and their proteins as well as CAK?Tyr402 protein expression were assayed by Western blot.?Results?(1) RNV was successfully induced with the use of a mice model of oxygen-induced retinopathy (OIR). In this model, mice are exposed to 75% oxygen tension from postnatal day (P) 7 to P12 and then transferred again to room air. At P14, retina in OIR group exhibited neovascularization, and manifested most obviously at P17. Changes in intracellular ROS levels in RNV model in mice had positive correlation to morphologic and functional changes in retina. (2) The highest VEGF mRNA and protein expression, CAK?mRNA and protein expression, and CAK?Tyr402/CAK?were found in C group at P12; The lowest VEGF, CAK?mRNA and protein expression were found in OIR group at P12, and predominantly increased at P14, at P17, VEGF, CAK?mRNA and protein expression had the highest levels. CAK?Tyr402/CAK?were statistically higher than in C group at P14 and P17; At P14, BVZ group showed significantly lower VEGF mRNA and protein expression than OIR group, and had no statistical difference when compared with C group. At P14, BVZ group also showed significantly lower CAK?mRNA and protein expression than OIR group, but significantly higher than C group. (3) FVS group and IBS group both showed statistically lower intracellular ROS levels, VEGF mRNA and protein expression, CAK?mRNA and protein and its Tyr402 protein expression than OIR group. Compared with either drug alone, combined Fluvastatin and Irbesartan treatment had greater decreasing levels mentioned above.?Conclusion?(1) Oxidative stress, VEGF and CAK?play important roles on RNV. (2) Bevacizumab down-regulates the expression of both VEGF and CAK?, but Bevacizumab has a more inhibiting effect on VEGF than CAK?. (3) Both Fluvastatin and Irbesartan have effective impact on decreasing of ROS, down-regulation expression of VEGF and CAK?, and attenuation of RNV. Dual therapy with Fluvastatin and Irbesartan produces an additive effect on attenuation of RNV. (4) CAK?exert its role on RNV in different signalling pathway, concentrating these pathway together. This may provide a potential therapeutic target for RNV. |
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