Abstract
Purpose: :
RPE cell survival is critically important in the normal eye and in diseases such as AMD and PVR. We have shown that Bcl–xL, an anti–apoptotic member of the Bcl–2 protein family, is among the most highly expressed survival factors in both cultured human RPE cells and in freshly isolated RPE cells from human donor eyes. Here, we determined the effect of Bcl–xL blockade on human RPE cell survival.
Methods: :
Cultured human RPE cells were transfected with various doses of the following modified, 2’–O–methoxyethoxy (MOE) oligonucleotides: Bcl–xL–antisense (ASO), Bcl–xL–mismatched control and Bcl–xL splice switching (SSO), which can convert Bcl–x pre–mRNA from the Bcl–xL mRNA splice variant into pro–apoptotic Bcl–xS. RNA and protein were harvested 1 day, 3 days, 5 days, and 7 days after transfection. Bcl–xL and Bcl–XS mRNA transcript levels were analyzed using gene–specific primers with real–time RT–PCR, and RT–PCR, respectively, and Bcl–xL protein expression was analyzed using western blot. Cell viability in serum–free medium was measured at the same time points using WST–1 assay.
Results: :
Following Bcl–xL–ASO and –SSO treatment, Bcl–xL mRNA and protein levels were reduced 70–80%, and 50–65%, respectively, compared to controls. Bcl–xS levels were significantly increased following Bcl–x– SSO treatment, while remaining unchanged by Bcl–xL–ASO treatment. By day 5, RPE cells transfected with Bcl–xL–ASOs showed significantly decreased viability compared to cells transfected with control oligonucleotides, an effect that was even greater by 7 days. The Bcl–x–SSO had an even more potent effect; cell viability was reduced after only one day; by day 7, less than 10% of the SS ASO–transfected cells were viable.
Conclusions: :
Bcl–xL plays an important role in human RPE cell survival. Treatment strategies that prevent conversion of Bcl–xL – to Bcl–xS may be useful to prevent RPE cell death normally and in AMD. Treatments to reduce Bcl–xL and enhance Bcl–xS may be a useful method to inhibit unwanted RPE cell proliferation in PVR.
Keywords: retinal pigment epithelium • apoptosis/cell death