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Morgan Rogers, Iris Navarro, Kristin Perkumas, R Rand Allingham, Pratap Challa, Craig Crosson, W Daniel Stamer; PEDF Effects on Outflow Facility. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2004.
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Preliminary data from our laboratory show that pigment epithelium derived factor (PEDF) increases transendothelial electrical resistance of human SC and porcine AAP endothelial monolayers in a time and dose-dependent manner. In this study, our goal was to determine PEDF levels in human aqueous humor, and the effect of PEDF on outflow function in enucleated mouse eyes.
Aqueous humor was obtained from consented patients during ocular surgery. Fresh samples were immediately placed on dry ice and kept frozen at -80C until an ELISA PEDF assay (Chemikine) was performed. Eyes from culled C57BL/6 mice were enucleated and cannulated for ex vivo perfusion using a computer-controlled perfusion system optimized for mouse eyes. Purified PEDF (1μg/ml) was perfused at four different pressures (4, 8, 15, 25 mmHg), measuring flow to determine outflow facility (slope of flow/pressure relationship). Data were compared to eyes perfused with vehicle alone (negative control) and two positive controls to establish range for detection of changes in outflow facility: dithia PGE-1 (10 nM) and sphingosine-1-phosphate (S1P, 5μM).
Eighteen human aqueous humor samples were examined and found to contain PEDF at levels ranging from 0.12-10.72 μg/mL (1.519 ± 2.492). We did not detect a relationship between PEDF level and gender, race or donor glaucoma status. We perfused a total of 27 C57BL/6 mouse eyes. Compared to vehicle-perfused controls, we observed an 86% increase in outflow facility for dithia PGE-1 (0.035 vs. 0.019 μl/min/mmHg), and a 20% decrease in outflow facility for S1P (0.017 vs. 0.022 μl/min/mmHg), similar to values previously reported. We perfused 7 eyes with PEDF and observed an average outflow facility of 0.018 μl/min/mmHg compared to 0.019 μl/min/mmHg for controls. Interestingly, there appeared to be two sets of responses within our data: 4 eyes having mean outflow facility of 0.010 ± 0.005 μl/min/mmHg, and the other 3 with mean of 0.028 ± 0.001 μl/min/mmHg.
PEDF is present in human aqueous humor at physiologically relevant concentrations. On average PEDF did not significantly affect outflow facility. However, the data set was bimodal with 4 eyes decreasing outflow facility by 50%, and the other 3 increasing outflow facility by 67%. We do not presently understand this phenomenon but have only tested one concentration of PEDF which was effective in vitro but may be too high ex vivo. We plan to perform a full concentration-response.
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