Epression Of Signal Transducer And Activator Transcription 3 In Experimental Choroidal Neovascuarization In Rat And The Effect On 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. CNV is one of the major causes of blindness that often involves the macula, leading to exudation, hemorrhage and scar foming and finally result in the loss of central vision. Researches on the mechanism of CNV have been a hot topic in the current field of ophthalmology, expecting to look for pathways preventing and curing CNV effectively.The exact causes of CNV are still unknown, an important initiating factor of CNV formation may be anoxyaemia. 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. A large array of stimulating factors impact the expression of VEGF, and the so involved molecular biology complicated much, creating a challenge for therapeutic methods targeting VEGF inhibition.While VEGF induces abound, only two major transcription activators have been identified for its promoter and can regulate the expression derectly: hypoxia inducible factor-1 (HIF-1) and signal transducer and activator of transcription 3 (STAT3). HIF-1 is an important oxygen-dependent transcriptional activator, which plays crucial roles in the angiogenesis. The up-regulation of VEGF expression in response to hypoxia occurs through HIF-1. STATs comprise a family of transcription factors which could be activated by many polypeptide ligand such as cytokine, growth factors. STATs contain a src homology 2 (SH2) domain and activated through tyrosine phosphorylation. Studies showed a significant increase of phosphorylated STAT3 in the pulmonary arterial smooth muscle cells after 2h exposure to hypoxia, implying that STAT3 could be involved in the reaction of cells by hypoxia. It has been found recently that the abnormal activated STAT3 plays an important role during the induction of tumor neovascularization. JAK2/STAT3 signal transduction pathway was activated in human renal cell under hypixa, regulate the expression of VEGF through the activation of HIF-1αprotein and promote the formation of neovasuclarization.Objective (1) To observe the location of phosphorylated STAT3 on the early stage of experimental CNV in rats induced by laser; (2) Set up the hypoxia model, and observe the change of STAT3 activation in cultured RPE cells under hypoxia; (3) To investigate the effect of JAK2/STAT3 signal transduction pathway on HIF-1αactivation and VEGF expression in cultured human RPE cells under hypoxia.Methods (1) The CNV model in rats was estabilished by laser, the location of phosphorylated STAT3 during the early stage of CNV was observed by immunofluorescence; (2) To set up the hypoxia model, human RPE cells were cultured in a special incubator containing volume fraction of 1%O2, 5% CO2 and 94% N2 for 0,1,3,6,12 and 24h. The expression of STAT3 and phosphorylated STAT3 were detected by RT-PCR and Western blot, the location of STAT3 in RPE cells was observed by immunofluorescence; (3) RPE cells were treated with/without 30μM JAK2 kinase inhibitor AG490 and cultured under hypoxia for 1, 3, 6, 12 and 24h, RPE cells proliferation activity were detected by flow cytometry (FCM), using RT-PCR and Western blot detected the expression of HIF-1αand VEGF, 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 phosphorylated STAT3 on 3d-CNV areas was observed and mainly located in proliferated and migrated RPE cells which involved in CNV ; (2) RT-PCR and Western blot detected that STAT3 mRNA and protein was poorly expressed in cultured human RPE cells. The expression of STAT3 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 STAT3 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 AG490 were significantly decreased comparing the control groups detected by FCM (P<0.05). RT-PCR showed the expression of HIF-1αmRNA andVEGF mRNA increased gradually and peaked at 6 h, the HIF-1αprotein peaked at 12h and decreased at 24h;while the AG490 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 AG490 treated group decreased significantly comparing the control groups (P<0.05).Conclusion (1) The phosphorylated STAT3 protein in RPE cells may be involved in the early stage of laser induced experimental CNV formation in rat; (2) STAT3 can be activated by hypoxia in hunman RPE cells; (3) JAK2/STAT3 pathway is an essential component of the signal transduction pathway to increase the VEGF expression in hypoxic RPE cells.