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J. Badhwar, P. C. Kothary, PhD, M. A. Del Monte, MD; The STAT Protein Inhibitor, AG 490, Inhibits Erythropoietin Production in Human Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5972.
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The human retinal pigment endothelium (hRPE) is a single layer of cells located between the photoreceptors and Bruch's membrane, which is normally quiescent after fetal life but may undergo proliferation and metaplasia under various pathological conditions. hRPE cells have also been implicated in retinal and choroidal neovascularization.
Erythropoietin (EPO), a glycoprotein produced in the kidneys and fetal liver, has been shown to have an angiogenic action similar to Vascular Endothelial Growth Factor, VEGF (Garcia 2009). Since EPO is a potent angiogenic factor that acts independently of VEGF during retinal angiogenesis in proliferative diabetic retinopathy, we investigated the effect of fetal bovine serum on EPO production in hRPE cells and role of the STAT signaling pathways in EPO production.
Primary hRPE cell cultures were established from human eyes obtained from the Michigan Eye Bank. HRPE cell proliferation was determined by triated thymidine incorporation (3h-thy) and cell viability by the trypan exclusion method (T). We conducted all studies in the presence and absence of a selective inhibitor of the STAT pathway, AG 490. Intracellular EPO synthesis was measured by immunoprecipitation using EPO specific antibody and intracellular EPO localized by immunohistochemical studies. Statistical Analysis was performed using Microsoft Excel, to determine Mean and SEM. Statistical differences between two groups of data were documented by the Student's 't' test.
Exposure to fetal bovine serum (FBS) (1-10%) stimulated hRPE cell proliferation in a dose dependent manner as determined by trypan blue exclusion and 3H-thymidine incorporation. FBS also stimulated 14C-EPO synthesis in a dose dependent manner. AG 490 (25 uM) decreased hRPE cell number as determined by T (0.75 ± 0.4787 vs. 1.75 ± 0.4787, cells/uL ± SEM, n=4, p<0.05) and 3H-thy incorporation (281.6 ± 140.8 vs 330.9 ± 165.4, CPM ± SEM, n=4, p<0.05). AG 490 also inhibited 14C-EPO synthesis (1678 ± 458.3 vs. 1934 ± 341.5, CPM ± SEM, n=6, p<0.05). Immuno-histochemical studies showed an increased immunoreactivity of EPO in presence of FBS (10%) and decreased immunoreactivity in presence of FBS (10%)+AG 490.
FBS stimulated synthesis of the angiogenic factor, erythropoietin, is mediated by the STAT signaling pathway and can be down-regulated by the STAT inhibitor AG 490. This information may be of therapeutic value in preventing or treating proliferative eye diseases.
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