The Inhibitive Effects Of Genistein On The Expression Of VEGF MRNA And Protein Induced By Hypoxia In ARPE-19 Cells

Neovascular diseases of the retina are one of the major causes of blindness in the world. Vision loss in pathological conditions such as diabetic retinopathy, retinopathy of prematurity, ischemic retinal-vein occlusion, and age-related macular degeneration is characterized by the extensive proliferation of new blood vessels in the retina. Hypoxia is the common stimulus of these pathologic states associated with uncontrolled angiogenesis. Recently, it is widely accepted by the researchers that a specific cytokine, vascular endothelial growth factor (VEGF) plays a primary role in angiogenic processes^[1-3]. It is an endothelial cell-specific mitogen which is stimulated by hypoxia and required for proliferation in some tumors. In ocular tissues, studies have demonstrated that VEGF production is increased by hypoxia in retinal cells, retinal pericytes. VEGF is expressed in the retina prior to the onset of neovascularization ina mouse model of proliferative retinopathy. Finally, there is a high correlation between VEGF expression and the retinal neovascularization associated with a variety of ocular diseases. Cultured human retinal cells such as pigment epithelial cells and pericytes secrete VEGF and increase VEGF gene and protein expression in response to hypoxia. Zhang et al ^[4,5] reported that hypoxia could induce the proliferation of RPE cells and the up-regulation of the expression of VEGF. Inhibition of the produce of VEGF in RPE cells can reduce neovascularization, than provide a new way for the therapy of Neovascular diseases of the retina.Current therapies such as panretinal photocoagulation and cryotherapy, are only partially effective and are destructive to the retina. Alternative, nondestructive modes of therapy could be of benefit to patients with these blindness-causing diseases. We found genistein, a naturally occurring isoflavone isolated from soybean^[6], provide a viable therapy for diseases characterized by retinal neovascularization . Recently it has been reported that genistein could inhibit neovascularization in a surgical model of choriocapillaris atrophy by surgical debriding the retinal pigment epithelium (RPE) in rabbits and in a model of retina ischemia in mice^[7]. Our previous^[18] study found that genistein could concentration-dependently inhibit VEGF protein expression induced by CoCl₂ and hypoxia in cultured rabbit retinal pigment epithelium(RPE) cells[]. Furthermore, genistein markedly inhibited the number of nucleiprotruding above the inner limiting membrane and the VEGF and HIF-1 a expression under relative hypoxia condition. Moreover, it has less toxicity than conventional chemical anti angiogenesis agents. Here, we observed the time course changes of VEGF expression induced by hypoxia and presented some evidence of the roles of genistein in the inhibition of VEGF, to study the mechanism of genistein in which it inhibit the expression of VEGF mRNA and protein induced by hypoxia in ARPE-19 cells.Objective:To study the influence of hypoxia on proliferation of ARPE-19 cells and expression of VEGF mRNA, investigate the inhibitive effects of genistein on hypoxia-induced VEGF mRNA and protein expression in ARPE-19 cells.Methods:ARPE-19 cells were exposed to hypoxia(5% CO2/95% N2) for 2, 12, 24, 36 hours, the proliferation of ARPE-19 cells was evaluated by [3(4,5 dimethylthiazole -2yl-2,5 -diphenyl) tetrazolium bromid, MTT] test; Then using RT-PCR to examine the expression of hypoxia-inducible VEGF mRNA. RPE cells pretreated with different concentration of genistein, PD98059, tyrphostin A25, were exposed to hypoxia for 2h and24h. The expression of VEGF mRNA was examined by RT-PCR, the expression of VEGF protein in cultured medium of RPE cells was detected by ELISA.Results:( 1 ) MTT: the OD value in the hypoxia groups were higher than that in the normal groups at all time points except 2 hours(P<0.05);RT-PCR: the expression of VEGF mRNA could be induced by hypoxia time-dependently, and reached its peak point at 24h.(2) Hypoxia for 2h: Hypoxia could up-regulate the expression of VEGF mRNA an...