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Haben Kefella, Shabnam Pakneshan, Tave van Zyl, Ron Adelman, Lawrence Rizzolo; Comparison of autophagy in hfRPE and ARPE-19 as two different models to study the pathogenesis of AMD. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1380.
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© ARVO (1962-2015); The Authors (2016-present)
Impaired autophagy has been implicated with drusen formation, lipofusion accumulation and Age-Related Macular degeneration (AMD). To explore autophagy in culture, we compared human fetal RPE (hfRPE) with the ARPE -19 cell line.
Secondary cultures of hfRPE were derived from 16-week human fetus. ARPE-19 cells, passge 25-27, originated from a 19-year old. Both cultures were grown to confluence and maintained for 4 weeks in low or no-serum media. Autophagosome formation was assessed morphologically by quantifying LC3-positive punctate structures and by using immunoblotting to monitor the conversion of LC3-I to LC3-II. Autophagic flux was inhibited using Spautin-1. RNA sequencing and qPCR was used to compare the expression of autophagy related genes.
ARPE-19 and hfRPE expressed similar levels of beclin-1 and LC3, two principle genes of the autophagy pathway. Spautin-1 inhibited autophagy flux with an LC-50 ~250 nM in ARPE-19, but doses greater than 50 nM were toxic for hfRPE.
The most differentiated cultures of RPE have been isolated from fetuses or young adults. Even though gene expression of key autophagy genes was similar, the greater sensitivity of hfRPE to specific inhibitors, like spautin, makes it a less attractive model compared to ARPE-19 to study this pathway.
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