Inhibitory Effects Of Rap1 On Experimental Choroidal Neovascularization

Objective: Choroidal neovascularization(CNV)is the common clinical manifestation and basic pathological changes of a variety of retinal diseases which leading to irreversible.The exact pathogenesis of CNV remains unclear,moreover there are still many problems need to be solved.Our recent research has shown that the expression of GTP-Rap1 in experimental CNV retinal pigment epithelial cells is lower than that in normal tissues,suggesting that Rap1 is related to the formation of CNV.On this basis,to determine the role of Rap1 in the formation of CNV,we have further observed the direct influence of GTP-Rap1 on CNV formation and the integrity of retinal pigment epithelium(RPE)barrier through activation of Rap1 in vivo.So the question is how GTP-Rap1 enhanced RPE barrier integrity and reduce the formation of CNV? Recent studies have found that GTP-Rap1 reduced reactive oxygen species(ROS)by copurified with the assembly of NADPH oxidase membrane subunit p22 phox.In addition,inhibition of NADPH oxidase-dependent ROS generation through intraperitoneal injection of apocynin also strengthens the RPE barrier integrity and reduce CNV.We hypothesize that GTP-Rap1 prevent CNV formation through inhibiting RPE barrier which suffers from ROS.To investigate the inhibitory influence of GTP-Rap1 on CNV,we established an experimental model of CNV by laser injury in the eyes of Brown Norway(BN)rats,and through intravitreal injection of 8cpt-cAMP to activate Rap1,apocynin as an antioxidant,observe whether the GTP-Rap1 reduces the formation of CNV by inhibiting oxidative stress pathways to strengthens RPE barrier integrity.Methods:1 Groups: Sixty 8-10 week healthy BN rats,which were randomly divided into three groups: laser model group,8CPT group which are injected8cpt-cAMP into intravitreal,APO group which are injected apocynin into abdominal cavity(20 rats in each group,40 eyeballs).2 CNV Model: Establish an experimental model of CNV by the Krypton laser(wavelength of 647 nm,a spot diameter of 200μm,power of 260 mW,exposure time of 0.05s).Around the optic disc,photocoagulation 9-10 points per eye avoiding retinal vascular.It proves that bruch membrane is punctured when cavitation bubbles appear.3 Inject Medicine: After laser model,the 8CPT group was injected with8cpt-cAMP respectively,and the injection volume was 1μl.The APO group was injected intraperitoneally with apocynin,0.1ml(10mg/kg/d),before the laser model is built.The observation is performed in 5days after laser.4 Real-Time PCR: five days after laser model,5 BN rats(10 eyes)were randomly chosen from three groups respectively.Remove the eyeballs after intraperitoneal anesthesia was performed.Then,detect p22phox/NOX4,Occludin mRNA transcription with Real-time PCR,comparing the change trend of the above groups.5 Western blot: five days after laser model,5 BN rats(10 eyes)were randomly chosen from three groups respectively.Remove the eyeballs after intraperitoneal anesthesia was performed.Then,detect p22phox/NOX4,Occludin proteins expression levels with Western blot,comparing the change trend of the above groups.6 DCFH-DA and flow cytometry: five days after laser model,5 BN rats(10 eyes)were randomly chosen from three groups respectively.Remove the eyeballs after intraperitoneal anesthesia was performed,fluorescence probe(DCFH-DA)was applied to RPE cells in CNV tissue.Then,the fluorescence intensity of RPE cells was observed by laser scanning confocal microscopy,and the level of ROS in RPE cells was detected by flow cytometry,comparing the change trend of the above groups.7 Choroid Flat Mounts result display: five days after laser model,5 BN rats(10 eyes)were randomly chosen from three groups respectively.After intraperitoneal anesthesia was performed,inject 1ml fluorescein isothioc-yanate in carotid artery.Then choroid flat mounts were carried out after removing the eyeballs to observe the formation of CNV with the laser scanning confocal microscope,and calculate the area of CNV to compare the change trend of the above groups.Results:1 Apocynin inhibits p22phox/NOX4 expression of RPE-Choroid-Sclera organization.Five days after laser model,p22phox/NOX4 mRNA and protein had lower expression in the APO group compared with the laser model group,and the difference was statistically significant(t=6.834,P<0.001;t=6.717,P<0.001).2 GTP-Rap1 inhibits p22phox/NOX4 expression of RPE-Choroid-Sclera organization.Five days after laser model,p22phox/NOX4 mRNA and protein had lower expression in the 8CPT group compared with the laser model group,and the difference was statistically significant(t=7.834,P<0.001;t=6.533,P<0.001).3 Apocynin increases Occludin expression of RPE-Choroid-Sclera organization.Five days after laser model,Occludin mRNA and protein expressed much more in the APO group compared with the laser model group,and the difference was statistically significant(t=-5.772,P<0.001;t=-6.763,P<0.001).4 GTP-Rap1 increases Occludin expression of RPE-Choroid-Sclera organization.Five days after laser model,Occludin mRNA and protein expressed much more in the APO group compared with the laser model group,and the difference was statistically significant(t=-5.797,P<0.001;t=-6.480,P<0.001).5 Apocynin inhibits ROS expression.Five days after laser model,the RPE cells expressed ROS in the laser model group is highly expressed compared with the APO group,and the difference was statistically significant(t=5.927,P<0.001).6 GTP-Rap1 inhibits ROS expression.Five days after laser model,the RPE cells expressed ROS in the laser model group is highly expressedcompared with the 8CPT group,and the difference was statistically significant(t=6.197,P<0.001).7 Apocynin inhibits CNV.Five days after laser model,the average CNV area of the APO group showed lower than the laser model group,from the perspective of statistical,this result is significant(t=8.601,P<0.001).8 GTP-Rap1 inhibits CNV.Five days after laser model,the average area of CNV of the 8CPT group showed a trend of decrease compared with the laser model group,from the perspective of statistical,this result is significant(t=7.034,P<0.001).Conclusion:GTP-Rap1 can enhance the integrity of RPE barrier and reduce the experimental CNV,the mechanism may related to the inhibition of ROS.