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Mary McGahan, Steven Nagar, Marilyn Lall; Hypoxia Affects Polarized Secretion of Amyloid Precursor Protein from Retinal Pigmented Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6352. doi: https://doi.org/.
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Amyloid precursor protein (APP) can be processed into different peptides, including amyloid beta (Aβ), which accumulates in plaques in Alzheimer's disease (AD) and in drusen in age-related macular degeneration. Activation of hypoxia-inducible factor increases APP processing to Aβ. Since APP alters retinal function, the purpose of this study was to determine how hypoxia affects the secretion of APP from retinal pigmented epithelial cells (RPE).
Canine RPE were grown as monolayers on either standard 6-well plates or Millicell inserts that allow access to the apical and basolateral compartments and development of polarized cell cultures . Experiments were performed when cells reached confluence, and in the case of Millicells, when cells were polarized as determined by development of high resistance (>100Ω) and polarized secretion of glutamate. Some cells were exposed to hypoxia (0.5% oxygen) in an hypoxia chamber for up to 48h. APP levels were determined by Western blotting of samples of cell-conditioned media and total cell lysates.
Medium from RPE cultured on standard plates contained APP and exposure to hypoxic conditions for up to 48h did not affect the APP secretion or the amount of APP in the cell lysates. In contrast, hypoxia dramatically affected directional secretion of APP in cells cultured on Millicell inserts. Under normoxic conditions, more APP was secreted in the basolateral than apical direction. After 48h of hypoxia, APP secretion in the basolateral direction was decreased, and importantly there was no detectable APP secretion to the apical bathing medium. There was a slight decrease in the amount of APP found in lysates from hypoxia-exposed cells compared to controls.
There are two significant observations from this study. First, hypoxia dramatically affects secretion of APP from RPE cells, reducing apical secretion to non-detectable amounts. It will be important to determine the mechanism underlying this effect. Second, cells grown as monolayers under conditions not allowing polarization showed no effect of hypoxia on APP secretion. This underscores the importance of using appropriate cell culture models for transport and secretion studies. Our model produces tight-junctional, polarized epithelial cells that better reflect in vivo conditions needed for physiologically relevant analysis of blood ocular-barrier function.
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