Abstract
Purpose:
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.
Methods:
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).
Results:
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.
Conclusions:
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.
Keywords: 427 aqueous •
735 trabecular meshwork •
543 growth factors/growth factor receptors