Expression Of Sp1 In Experimental Choroidal Neovascuarization In Rat And The Role Of Sp1 In Transcription Control Of Vascular Endothelial Growth Factor In The Cultured Human Retinal Pigment Epithelial Cells Under Hypoxia

Background Choroidal neovascularization (CNV) occurs in multiple ophthalmic diseases such as age-related macular degeneration (AMD), ocular histoplasmosis syndrome (OHS), pathological myopia, ocular trauma and so on.,which is is now known the leading cause of vision loss in age related macular degeneration (AMD), while its pathogenesis is still poorly understood. Retinal pigment epithelium (RPE) cells are known to quickly respond and adapt to environmental stresses such as ischemia and metabolic changes by expressing a number of various genes. In the early stages of CNV development, RPE produce cytokines and growth factors promoting CNV development.The underlying mechanism of CNV is multifactorial and complex. Growth factors and cell adhesion molecules have been implicated in CNV, and anoxyaemia may be an important initiating factor of CNV formation. The change of local microenvironment induces choriocapillary occlusion, atrophia and fibrosis, leads to insufficiency of choroids blood supply, results in retinal anoxyaemia, thus stimulate retinal pigment epithelium (RPE) division and proliferation, secreting various cytokines such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and so on, then draws the choriocapillary growth compensatorily toward outer layer of retina, and the CNV formed finally.VEGF is upregulated by hypoxia, it is the most powerful pro-angiogenic factor found till now, and also an important cytokinin. RPE is an major cell component of CNV, the expression of VEGF in RPE cells under hypoxia may be a key link of CNV formation. Recent work from several laboratories points out the importance of Sp1 in influencing distinct steps of the angiogenic response and suggests a critical role of Sp1 in tumor angiogenesis. But to our knowledge, no study investigating the effect of Sp1 on CNV formation has been reported previously.Objective (1) To observe the expression and location of Sp1on the early stage of experimental CNV in rats induced by laser; (2) Set up the hypoxia model, and observe the expression of Sp1 in cultured RPE cells under hypoxia; (3) To investigate the effect of Sp1 signal transduction pathway on VEGF expression in cultured human RPE cells under hypoxia using specific inhibitor Mithramycin A.Methods (1) The CNV model in rats was estabilished by laser, the location of phosphorylated Sp1 during the early stage of CNV was observed by immunofluorescence; (2) Set up the hypoxia model using Cobalt Chloride, and observe the expression of Sp1 in cultured RPE cells under hypoxia. The expression of Sp1 were detected by RT-PCR and Western blot, the location of Sp1 in RPE cells was observed by immunofluorescence; (3) RPE cells were treated with/without 200nM specific inhibitor Mithramycin A and cultured under hypoxia for 2, 4, 8, 12 and 24h, RPE cells proliferation activity were detected by flow cytometry (FCM), using RT-PCR detected the expression of VEGF mRNA, the amounts of VEGF in the RPE-conditioned supernatant were measured using enzyme linked immunosorbent assay (ELISA) kits.Results (1) A strong positive staining for Sp1 was observed and mainly located in proliferated and migrated RPE cells which involved in CNV ; (2) RT-PCR and Western blot detected that Sp1mRNA and protein was poorly expressed in cultured human RPE cells. The expression of Sp1 increased gradually and peaked at 12h in the cultured RPE cells treated with hypoxia, then decreased but still higher than normoxia at 24h. The fluorescence of Sp1 protein mainly located in the cytoplasm in the cultured RPE cells under normoxic conditions. There was a decrease in green fluorescence intensity within the cytoplasm while the fluorescence intensity in the nucleus increased under hypoxic conditions; (3) The proliferation activity of RPE cells in the groups treated with MithramycinA were significantly decreased comparing the control groups detected by FCM (P<0.05). RT-PCR showed the expression of VEGF mRNA increased gradually and peaked at 12 h,;while the MithramycinA treated groups were suppressed obviously comparing the control groups; The amount of VEGF in RPE cell supernatant was significantly increased in time-depentent manner under hypoxia, and the amount of VEGF in conditioned medium of MithramycinA treated group decreased significantly comparing the control groups (P<0.05).Conclusion (1) The Sp1 protein in RPE cells may be involved in the early stage of laser induced experimental CNV formation in rat; (2)Hypoxia can induce the high expression of Sp1 in cultured RPE cells.(3) Sp1 can lays an important role of increasing VEGF expression in hypoxic RPE cells.