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Nandita Anand, Piyush Kothary, Monte Del Monte; Quercetin Inhibits H2O2 Stimulated PEDF and FGF2 Synthesis in hRPE Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4588.
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© ARVO (1962-2015); The Authors (2016-present)
Age related macular degeneration (AMD) is a major cause of blindness. Proliferation and damage of the human retinal pigment epithelial (hRPE) cells is involved in the pathogenesis of AMD. Oxidative stress induces in hRPE cell death and stimulates the synthesis of angiogenic factors in hRPE cells. We studied the effect of quercetin (Q), a flavonoid that protects cells from oxidative stress, on the synthesis of the angiogenic factor, fibroblast growth factor 2 (FGF2), and the anti-angiogenic factor, pigment epithelial derived factor (PEDF) in hRPE cells.
hRPE cells were cultured from eyes obtained from the Michigan Eye Bank.Cellular proliferation in the presence of increasing concentrations of fetal bovine serum (FBS) was measured by 3[H] thymidine incorporation. hRPE cell viability was measured in the presence of increasing concentrations of FBS and H2O2 by the trypan blue exclusion method (T).The effect of (Q) on hRPE cell viability was also measured in presence of H2O2 by (T).The effect of various concentrations of H2O2 on intracellular synthesis of PEDF and FGF2 in presence and absence of (Q) was measured by immunoprecipitating 14C-methionine-PEDF (14C Met-PEDF) and 14C-methionine-FGF2 (14C Met-FGF2) and immunocytochemical (IC) analysis.
FBS (0-10%) stimulated cell proliferation and cell viability in cultured hRPE cells. H2O2 (0-0.5mM) decreased cell viability (T) in a dose dependent manner. The addition of (Q) (50μM) to 0.5mM H2O2 treated cells increased hRPE cell viability(130,000 ± 48,989 vs. 40,625± 28,252, n=8, cells±SEM, p<0.05). H2O2 exposure increased both (14C Met-PEDF) and (14C Met-FGF2) synthesis in a dose dependent manner. (Q) inhibited H2O2 stimulated intracellular (14C Met-PEDF) synthesis in hRPE cells(441.81 ±198.54 vs. 269.016± 96.09, n=10, CPM±SEM, p<0.05). (Q) also inhibited H2O2 stimulated (14C Met-FGF2)(147.8±9.01 vs.100.92±23.38, n= 5, CPM±SEM, p <0.05). Phase contrast microscopy using DAPI or 4',6-diamidino-2-phenylindole fluorescent stain showed that H2O2 damages hRPE cell morphology and the addition of (Q) protects the cells from damage. (IC) studies confirmed inhibition of H2O2 stimulated FGF2 expression by (Q) in hRPE cells.
We concluded that (Q) inhibits H2O2 stimulated PEDF and FGF2 synthesis in hRPE cells.(Q) may rescue hRPE cells by inducing equilibrium in the synthesis of PEDF and FGF2 in hRPE cells.(Q) may have therapeutic value in the treatment of AMD.
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