Expression of Rap1, guanosine triphosphate Rap1, vascular endothelial growth factor and β-catenin in experimental choroidal neovascularization_Chinese Journal of Ocular Fundus Diseases

Authors：

Wang Xin ,  Shang Qingli , Ma Jingxue , Hao Yuhua , Yao Huijuan , Li Jiajia

Department of Ophthalmology, Affiliated Second Hospital of Hebei Medical College, Shijiazhuang 050000, China;

Corresponding author：

Shang Qingli, Email: qinglishang2013@sina.cn

Keywords：

Choroidal neovascularization/treatment; rap1 GTP-binding proteins; Vascular endothelial growth factors; beta Catenin

DOI：

10.3760/cma.j.issn.1005-1015.2018.05.013

Video：

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ObjectiveTo observe the expression of Rap1, guanosine triphosphate-Rap1 (GTP-Rap1), vascular endothelial growth factor (VEGF) and β-catenin in experimental choroidal neovascularization (CNV).MethodsForty-two brown Norwegian rats were randomly divided into a blank control group (7 rats) and a model group (35 rats). Both eyes were enrolled. The CNV model was established by holmium ion laser photocoagulation in the model group. At 3, 7, 14, 21, and 28 days after photocoagulation, fluorescein fundus angiography (FFA) and choroidal vascular smear were performed to observe the degree of fluorescein leakage and CNV area in rats; Western blot and real-time quantitative polymerase chain reaction (RT-PCR) were used to detect the expression of Rap1, GTP-Rap1, VEGF, β-catenin and mRNA in CNV.ResultsThe results of FFA examination showed that a large disc-shaped fluorescein leaked in the photo-condensation spot 14 days after photocoagulation. Laser confocal microscopy showed that compared with 7 days after photocoagulation, CNV area increased at 14, 21, 28 days after photocoagulation, and the difference were statistically significant (t=3.725, 5.532, 3.605;P＜0.05). Western blot showed that there was no significant difference in the relative expression of Rap1 protein in CNV at different time points after photocoagulation between the two groups (P=0.156). Compared with the blank control group, the relative expression of GTP-Rap1 protein was significantly decreased, the relative expression of VEGF and β-catenin protein were significantly increased in the model group (P=0.000). The results of RT-PCR showed that there was no significant difference in the relative expression of Rap1 mRNA at different time points after photocoagulation between the two groups (P=0.645), but there were significant difference in the relative expression of β-catenin mRNA (P=0.000). At 7, 14, 21 and 28 days after photocoagulation, there were significant difference in the relative expression of GTP-Rap1 and VEGF mRNA between the two groups (P=0.000).ConclusionsThe expression of GTP-Rap1 in experimental CNV is significantly lower than that in normal rats.

Citation： Wang Xin, Shang Qingli, Ma Jingxue, Hao Yuhua, Yao Huijuan, Li Jiajia. Expression of Rap1, guanosine triphosphate Rap1, vascular endothelial growth factor and β-catenin in experimental choroidal neovascularization. Chinese Journal of Ocular Fundus Diseases, 2018, 34(5): 475-480. doi: 10.3760/cma.j.issn.1005-1015.2018.05.013 Copy

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1. Mikačić I, Bosnar D. Intravitreal bevacizumab and cardiovascular risk in patients with age-related macular degeneration: systematic review and meta-analysis of randomized controlled trials and observational studies[J]. Drug Saf, 2016, 39(6): 517-541. DOI: 10.1007/s40264-016-0408-y.
2. 赵世红, 何守志.氪激光诱导的大鼠脉络膜新生血管模型研究[J]. 中华眼科杂志, 2003, 39(5): 298-302. DOI: 10.3760/j:issn:0412-4081.2003.05.012.Zhao SH, He SZ.Study on the experimental model of krypton laser-induced choroidal neovascularization in the rats[J]. Chin J Ophthalmol, 2003, 39(5): 298-302. DOI: 10.3760/j:issn:0412-4081.2003.05.012.
3. Maguire MG, Daniel E, Shah AR, et al. Incidence of choroidal neovascularization in the fellow eye in the comparison of age-related macular degeneration treatments trials[J]. Ophthalmology, 2013, 120(10): 2035-2041. DOI: 10.1016/j.ophtha.2013.03.017.
4. Velez-Montoya R, Oliver SC, Olson JL, et al. Current knowledge and trends in age-related macular degeneration: today's and future treatments. Retina, 2013, 33(8): 1487-1502. DOI: 10.1097/IAE.0b013e318271f265.
5. Wittchen ES, Nishimura E, McCloskey M, et al. Rap1 GTPase activation and barrier enhancement in rpe inhibits choroidal neovascularization in vivo[J/OL].PLoS One, 2013, 8(9): 73070[2013-09-10]. https://doi.org/10.1371/journal.pone.0073070. DOI: 10.1371/journal.pone.0073070.
6. Wang H, Jiang Y, Shi D, et al. Activation of Rap1 inhibits NADPH oxidase-dependent ROS generation in retinal pigment epithelium and reduces choroidal neovascularization[J]. FASEB J, 2014, 28(1): 265-274. DOI: 10.1096/fj.13-240028.
7. Chakravarthy U, Harding SP, Rogers CA, et al. Alternative treatments to inhibit VEGF in age-related choroidal neovascularisation: 2-year findings of the IVAN randomised controlled trial[J]. Lancet, 2013, 382(9900): 1258-1267. DOI: 10.1016/S0140-6736(13)61501-9.
8. Ahmadi MA, Lim JI. Pharmacotherapy of age-related macular degeneration[M].Expert Opin Pharmacother, 2008, 9(17): 3045-3052. DOI: 10.1517/14656560802473480.
9. Burke JM, Hong J.Fate of E-cadherin in early RPE cultures: transient accumulation of truncated peptides at nonjunctional sites[J]. Invest Ophthalmol Vis Sci, 2006, 47(8): 3635-3643. DOI: 10.1167/iovs.06-0104.
10. McKay BS, Irving PE, Skumatz CM, et al. Cell-cell adhesion molecules and the development of an epithelial phenotype in cultured human retinal pigment epithelial cells[J]. Exp Eye Res, 1997, 65(5): 661-671. DOI: 10.1006/exer.1997.0374.
11. Youn YH, Hong J, Burke JM.Cell phenotype in normal epithelial cell lines with high endogenous N-cadherin: comparison of RPE to an MDCK subclone[J]. Invest Ophthalmol Vis Sci, 2006, 47(6): 2675-2685. DOI: 10.1167/iovs.05-1335.
12. Palma-Nicolás JP, López-Colomé AM.Thrombin induces slug-mediated E-cadherin transcriptional repression and the parallel up-regulation of N-cadherin by a transcription- independent mechanism in RPE cells[J]. J Cell Physiol, 2013, 228(3): 581-589. DOI: 10.1002/jcp.24165.
13. Gullapalli VK, Sugino IK, Van Patten Y, et al. Impaired RPE survival on aged submacular human Bruch's membrane[J]. Exp Eye Res, 2005, 80(2): 235-248. DOI: 10.1016/j.exer.2004.09.006.
14. Lee DK, Nathan Grantham R, Trachte AL, et al. Activation of the canonical Wnt/beta-catenin pathway enhances monocyte adhesion to endothelial cells[J]. Biochem Biophys Res Commun, 2006, 347(1): 109-116. DOI: 10.1016/j.bbrc.2006.06.082.
15. Clevers H, Nusse R.Wnt/β-catenin signaling and disease[J]. Cell, 2012, 149(6): 1192-1205. DOI: 10.1016/j.cell.2012.05.012.
16. Voronkov A, Krauss S.Wnt/beta-catenin signaling and small molecule inhibitors[J]. Curr Pharm Des, 2013, 19(4): 634-664.
17. Monga SP.β-catenin signaling and roles in liver homeostasis, injury, and tumorigenesis[J]. Gastroenterology, 2015, 148(7): 1294-1310. DOI: 10.1053/j.gastro.2015.02.056.
18. Schneider AJ, Branam AM, Peterson RE.Intersection of AHR and Wnt signaling in development, health, and disease[J]. Int J Mol Sci, 2014, 15(10): 17852-17885. DOI: 10.3390/ijms151017852.
19. Hu Y, Chen Y, Lin M, et al. Pathogenic role of the Wnt signaling pathway activation in laser-induced choroidal neovascularization[J]. Invest Ophthalmol Vis Sci, 2013, 54(1): 141-154. DOI: 10.1167/iovs.12-10281.
20. Genead MA, Mcanany JJ, Fishman GA. Topical dorzolamide for treatment of cystoid macular edema in patients with choroideremia[J]. Retina, 2012, 32(4): 826-833. DOI: 10.1097/IAE.0b013e3182215ae9.
21. Adijanto J, Banzon T, Jalickee S, et al. CO2-induced ion and fluid transport in human retinal pigment epithelium[J]. J Gen Physiol, 2011, 133(6): 603-622. DOI: 10.1085/jgp.200810169.
22. Wang H, Han X, Bretz CA, et al. Retinal pigment epithelial cell expression of active Rap 1a by scAAV2 inhibits choroidal neovascularization[J]. Mol Ther Methods Clin Dev, 2016, 3: 16056. DOI: 10.1038/mtm.2016.56.

Chinese Journal of Ocular Fundus Diseases

Expression of Rap1, guanosine triphosphate Rap1, vascular endothelial growth factor and β-catenin in experimental choroidal neovascularization

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