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Piyush C Kothary, Allen Lee, Eric Frontera, Priyanka Varma, Danna Bismar, Monte A Del Monte; INSULIN LIKE GROWTH FACTOR 1 INDUCTION OF HYPOXIA-INDUCIBLE FACTOR MEDIATED VASCULAR ENDOTHELIAL GROWTH FACTOR SYNTHESIS IS DEPENDENT ON MAP KINASE ACTIVITY. Invest. Ophthalmol. Vis. Sci. 2014;55(13):359. doi: https://doi.org/.
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Human retinal pigment epithelium (hRPE) cells appear to play a role in the pathogenesis of proliferative diabetic retinopathy (PDR). Hypoxia-inducible factor (HIF) is a heterodimer made of HIF1 alpha (HIF1a) and HIF1 beta (HIF1b). We have shown that HIF1a mediates the angiogenic factor, vascular endothelial growth factor (VEGF) synthesis in cultured hRPE cells. However, very little is known about the role of HIF1b in the regulation of VEGF synthesis in hRPE cells. Since IGF1 also plays an important role in pathogenesis of diabetes and proliferative diabetic retinopathy (PDR), we investigated the effect of IGF1 on HIF1a, HIF1b and VEGF synthesis in hRPE cells.
hRPE cultures were established from normal human eyes. Cell proliferation and viability were quantitated by 3H-thymidine (3H-thy) incorporation and by the trypan blue exclusion (T) method. 14C-VEGF, 14C-HIF1 alpha and 14C-HIF1b production were measured by immunoprecipitation and immunohistostochemistry. PD98059 (PD), an inhibitor of MAPK was used to study the mechanism. Statistical significance was determined by Student “t” test.
Increasing concentrations of fetal bovine serum and IGF1 stimulated hRPE proliferation in culture as determined by T and 3H-thy. IGF1 (0-50 ng/ml) also stimulated 14C-VEGF, 14C-HIFa and 14C-HIFb synthesis in a dose-dependent manner. PD98059 inhibited IGF1 stimulated 14C-VEGF synthesis (646.73±165.61 vs. 1184.40±247.95, CPM±SEM, p<0.05, n=4), 14C-HIFa synthesis (249.18±60.14 vs. 383.81±96.43, CPM±SEM, p<0.05, n=5) and 14C-HIF1b synthesis (87.78±18.36 vs. 166.49±25.96, CPM±SEM, p<0.05, n=4). Immuno-histochemical studies confirmed that IGF1 increases VEGF, HIF1a and HIF1b protiens when compared to controls. Linear curve analysis showed that IGF1 stimulation of HIFa and HIFb were superimposable. VEGF synthesis was less than HIF1a and HIF1b synthesis at a lower concentration of IGF1 but was almost same as that of HIF1a and HIF1b when exposed to higher concentrations of IGF1.
We conclude that IGF1 stimulated VEGF synthesis in hRPE cells. In addition, IGF-1 induced HIF1a and HIF1b-mediated VEGF expression is dependent on MAP kinase signaling in these cells. Therefore an inhibitor of MAP kinase signaling may be of therapeutic value in PDR or proliferative vitreoretinopathy.
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