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Carolyn Cai, Lucian V. Del Priore; Bruch's Membrane Aging Reduces Autophagic Activity in Retinal Pigment Epithelium: Implications for Age-related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2011;52(14):874.
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We have reported age-related alterations in Bruch's membrane decreases phagocytic function in retinal pigment epithelium (RPE). Autophagy is a cellular process for the removal of damaged or abnormal cellular components. Herein we investigate if aging of human Bruch’s membrane affect autophagic activity in RPE.
Human ARPE19 cells were cultured to confluence for up to 3 weeks on human Bruch's membrane explants harvested from young (age < 50) or older (age > 70) human eye bank eyes as previously described (Tezel TH, Del Priore LV.IOVS 1999;40:767-774). Invitrogen's LC-3 fluorescence protein (FP) Baclovirus was transduced into ARPE19. Fluorescence microscope image densitometry was obtained using images taken from 4X lens exposed for 3 seconds, 5 images per culture explant (center, four corners). Autophogic activities are correlated with the intensity of LC-3 GFP fluorescence; LC-3 antibody Western blots of ARPE19 cell lysate also were used to monitor cell autophagy. NIH ImageJ software was used for densitometry quantification.
ARPE cells reach confluence less in culture on older Bruch’s membrane. 72 hour after transduction LC3 fluorescence protein can be visualized with fluorescence microscope for RPE cells cultured on young Bruch’s membrane surrounding the cell nucleus, thus suggesting autophagic activity. Immunofluorescent images demonstrated that LC-3-GFP was scattered around in cell cytosol of RPE maintained on older Bruch's membrane. Quantitative fluorescence microscope image densitometry analysis shows 11% less fluorescence intensity in RPE cells on older versus younger Bruch’s membrane. LC-3 western blot analysis shows more activated form of LC-3 (lower molecular weight) in RPE cells cultured on younger Bruch’s membrane.
The results suggest that Bruch’s membrane aging reduces autophagic activity in RPE. Aging of human Bruch's membrane, which is associated with numerous changes within Bruch’s membrane, may adversely affect the ability of the RPE to maintain healthy intracellular homeostasis. The significance of these changes for the pathogenesis of age-related macular degeneration remains to be elucidated.
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