Expressions Of Rac1 And HIF-1α In Cultured Human Retinal Pigment Epithelial Cells Under Hypoxia And Choroidal Neovascularization Induced By Laser In Mice

Background Choroidal neovascularization (CNV) arises from the choriocapillaries through Bruch's membrane into the subretinal space. It has been described as a cause of visual loss in more than thirty ocular diseases, including age-related macular degeneration (AMD), pathologic myopia, ocular histoplasmosis, angioid streaks, trauma, multifocal choroiditis, and polypoidal choroidal vasculopathy (PCV). These complications are associated with the atrophy and senescence of retinal pigment epithelium (RPE) cells and microfractures in Bruch's membrane, as well as exudation of fluid and hemorrhage into the subretinal space. CNV is believed to occur from transmigration of choriocapillaries endothelial cells (CEC) across the retinal pigment epithelium (RPE) into the neurosensory retina. The transform of choroids circulation and the incrassation of Bruch's membranein the CNV patients could lead to hypoxia condition. Studies show that HIF-1 is an oxygen-dependent transcriptional activator, which plays crucial roles in the angiogenesis. Recent investigations have revealed that Rac1 is also an important regulator under hypoxia, which modulate the activation of hypoxia-inducible factor-1α(HIF-1α) and that Rac1 maybe play an important role in modulation of many factors in angiogenesis. The studies on Rac1 in the past are more focused on tumors. Rac1 protein almost participates in the every stages of tumors, such as tumor generation,invasion,metastasis and the level of malignant tumor and so on. In angiogenesis, Rac1 regulates extracellular matrix and the Chemotaxis of endothelial cells (EC) linked to overexpression of vascular endothelial growth factor (VEGF), which promotes choroidal EC migration and proliferation. It revealed Rac1 is associated with neovascularization. However, few studies have been reported in the relation between Rac1 and the underlying mechanism of CNV yet at home and abroad.Purpose: To determine the expression of Rac1 and HIF-1αin cultured human retinal pigment epithelial (hRPE) cells under hypoxia and CNV model in mice. Methods: (1) The human RPE cells were cultured in DMEM containing 200μmol.L-1 CoCl2 for 0.5,1,2,4,8,12 and 24 h. The hRPE cells were cultured under normoxia as controls. The expression of Rac1 was measured in both controls and hypoxia-induced hRPE cells by RT-PCR,immunocytochemical staining and Western blot, the expression of HIF-1αmeasured by immunocytochemical staining. (2) CNV mice models were induced by 532 nm laser and the eyeballs were taken out at the 1 st,3 rd,7 th,14 th and 28 th day respectively after fluoresceinangiography was performed . The expression of Rac1 and HIF-1αin CNV tissues was detected by immunohistochemistry.Results: (1) RT-PCR result showed the expression of Rac1 mRNA was positive and was time-dependently increased in the hypoxia group with the peak at 4 h. Comparing with control group, there is a significant difference(P<0.05);The Western blotting analysis showed that the level of expression of Rac1 had a result similar to that of RT-PCR. But Rac1 protein reached the top at 8h under hypoxia;Rac1 and HIF-1αin hRPE are both negative under normoxia.The expression of Rac1 was found in the hRPE cells as early as 0.5 h after induction of hypoxia with immunocytochemical staining, and it mainly localized in the cytoplasm. There was a time-dependently increase in the expression of Rac1 with the peak at 8 h, and declined at 12 h after exposure under hypoxia. The level of expression of Rac1 in the cells cultured under hypoxia showed a significant difference when compared to that in the controls (P<0.05). The expression of HIF-1αmainly localized in the cytoplasm and nucleus, reached the top at 12 h and decreased at hypoxia for 24 h. It also showed significant difference when compared to that in the controls (P<0.05). There was a significantly linear relationship between Rac1 and HIF-1αaccording to the linear regression analysis (P<0.001). (2) Rac1 and HIF-1αwere both significantly expressed in experimentalmouse CNV tissues induced by 532nm laser. Their expression both increased during the earlier period after photocoagulation and achieved the peak at 3 and 7 days after laser treatment. The ratio of Rac1 positive expression was 34.58% and 53.35% respectively, and the positive expression of HIF-1αwas 33.05% and 50.35%. Two weeks after laser treatment, the expression of Rac1 and HIF-1αboth decreased, which were about 25.50% and 31.97% respectively. There was obvious correlation between the expression of Rac1 and HIF-1α(r=0.943, P =0.016).Conclusion: The expression of Rac1 in hRPE cells can be up-regulated in response to hypoxia and they are both significantly expressed in experimental CNV tissues. There was significantly positive association between the level of Rac1 and HIF-1α. It revealed that Rac1 and HIF-1αare closed correlated with the Choroidal neovascularization.Innovational points of this study: Rac1 and HIF-1αplay important roles in angeogensis. There was significantly positive association between the level of Rac1 and HIF-1αin the early stage of the CNV. To the best of our knowledge, similar findings have not been reported yet at home and abroad.