An Experimental Study On The Inhibition Effect Of Rap1 On Choroidal Neovascularization

Objective : Choroidal neovascularization is the common and basic pathological change of a variety of ocular fundus diseases.Because of involving the macular region of the retina,it often causes repeated hemorrhage,exudation and scarring of posterior choroid and retina,and seriously affects the patients’ central vision.The integrity of the retinal pigment epithelium-Bruch’s membrane-choriocapillaris complex is closely related to the occurrence and development of CNV.In the CNV related diseases,loss of RPE barrier integrity occurs before migration,inflammation and other pathological processes.Previous studys have found that a small molecular G protein,Rap1,which belongs to the Ras superfamily,plays an important role in regulating cell movement and junction formation.It is involved in the regulation of tight junctions and adherens junctions between epithelial cells and endothelial cells,so as to enhance the integrity of the barrier function.Our previous study has confirmed that compared with normal tissue,GTP-Rap1,the activated form of Rap1,decreased in experimental CNV,showing a correlation between Rap1 and CNV and suggesting that enhancing Rap1 activity may inhibit CNV formation by protect the RPE barrier function.However,the specific effects of Rap1 on CNV and the integrity of RPE barrier haven’t been observed by in vivo activation of Rap1.Thus,we haven’t determine the direct role of Rap1 in CNV occurrence and development.This study established laser photocoagulation-induced CNV models of BN rats,and activated Rap1 by intravitreal injection of 8cpt-cAMP.Then the development of CNV and expression level of related factors in CNV tissue were detected,so as to investigate the specific effect of Rap1 on the RPE barrier function and CNV development.Methods:1 Seventy 8-10 week healthy male BN rats,whose anterior segment and ocular fundus were normal,both by slit lamp and direct ophthalmoscope examination,were randomly divided into experimental group and blank control group(35 rats in each group,70 eyes).The laser-induced CNV model was carried out as described here.Krypton laser photocoagulation(wavelength of 647 nm,spot diameter of 200μm,power of 260 mW,exposure time of 0.05s)was performed around the optic disc.Laser spots(9-10 points per eye)were applied 2-3 disc diameters from the optic nerve,avoiding major vessels.Disruption of Bruch’s membrane was confirmed by the appearance of a cavitation bubble.2 After laser injury,a 1-μl intravitreal injection of 8cpt-cAMP or control PBS was delivered.The observation time is 3d,7d,14 d,21d,28 d after laser photocoagulation.3 At 14 d after laser teratment,5 rats were randomly selected from each group,and after intraperitoneal anesthesia,an injection of 1ml fluorescein isothiocyanate-dextran was carried in carotid artery.Then the eyeballs were removed,and choroid flat mounts were performed to observe CNV formation under laser scanning confocal microscope.The area of CNV was measured and compared between the two groups.4 15 rats were randomly selected from each group,and eyeballs were removed at 3d,7d,14 d,21d,28 d respectively(3 rats for each observation time,6 eyes)after intraperitoneal anesthesia.Real-Time PCR was performed to detect and compare the mRNA transcription levels of GTP-Rap1,ZO-1 and VEGF genes in CNV tissues and normal tissues,and to observe the change trend of the above indicators.5 The remaining 15 rats were selected from each group,and eyeballs were removed at 3d,7d,14 d,21d,28 d respectively(3 rats for each observation time,6 eyes)after intraperitoneal anesthesia.Western blot was performed to detect and compare the expression levels of GTP-Rap1,ZO-1 and VEGF proteins in CNV tissues and normal tissues,and to observe the change trend of the above indicators.6 The experimental data were statistically analyzed with SPSS22.0 software package.Results:1 Choroid flat mounts:Two weeks after laser treatment,high fluorescence CNV network formed irregularly at laser spots.The average area of CNV in experimental group was bigger than control group,and the difference was statistically significant(P<0.001).2 Real-Time PCR:GTP-Rap1 and ZO-1 mRNA increased and VEGF mRNA decreased with observation time in 8cpt-c AMP group.GTP-Rap1 mRNA showed a gradual decreasing trend and VEGF mRNA increased with observation time in PBS group.At each time point,GTP-Rap1 mRNA expressed much more and VEGF mRNA had lower expression in experimental group compared with control,and the difference was statistically significant since day 7 after laser injury(P<0.001).On the 14 d after laser treatment,the relative expression of ZO-1 mRNA in the experimental group was much higher compared with the control group,and the difference was statistically significant(P<0.001).3 Western blot:GTP-Rap1 and ZO-1 increased gradually and VEGF decreased gradually with observation time in 8cpt-cAMP group.GTP-Rap1 protein expression showed a gradual decreasing trend and VEGF protein expression increased with observation time in PBS group.At each time point,GTP-Rap1 protein expressed much more and VEGF mRNA had lower expression in experimental group compared with control,and the difference was statistically significant since day 7 after laser injury(P<0.001).On the 14 d after laser treatment,the relative expression of ZO-1 protein in the experimental group was much higher compared with the control group,and the difference was statistically significant(P<0.001).Conclusion:1 Activating Rap1 can effectively strengthen the tight junctions between cells,and enhance the integrity of RPE barrier further.2 Activation of Rap1 can significantly inhibit the formation of experimental CNV by RPE barrier enhancement,and reducing the expression of VEGF.3 This experiment provides a new idea for the treatment of CNV——Activation of Rap1 is a promising and new way to treat CNV,which provides a reliable theoretical basis for the future experimental study.