Abstract
Purpose:
Vascular endothelial growth factor (VEGF) is a secreted protein with a variety of functions including angiogenesis, vasodilation, and increased membrane permeability. Intravitreal anti-VEGF injection is the current mainstay treatment for many retinal diseases. Of recent concern, anti-VEGF injections have resulted in an alarming increase in intraocular pressure (IOP) in a subset of patients, the mechanism of which is unknown. Currently, there is little understanding of VEGF and its roles in the outflow tract and glaucoma. In this study, we characterize endogenous and stress-induced VEGF secretion in the outflow tract, and we test the hypothesis that VEGF increases outflow facility in vivo.
Methods:
Human trabecular meshwork (TM) and Schlemm’s canal (SC) cells were isolated and grown in culture. Endogenous VEGF levels (isoform 165) were quantified by ELISA from cell supernatants. TM cells were plated onto flexible membranes, and cyclic mechanical stretch (16% elongation, 1 cycle/s) was applied for 24 hours (Flexcell); or TM cells were treated with 100 nM dexamethasone (dex) for up to 7 days. To determine the effect of VEGF on SC cell permeability, SC cells were grown to confluence on transwell filters, and transendothelial electrical resistance (TEER) was measured in response to VEGF (25, 100 ng/ml). To study the effect of VEGF on outflow facility, VEGF (100 ng/ml) or PBS was infused into the anterior chambers of living mice. Flow was measured at fixed pressures (15, 25, 35 mmHg), and conventional outflow facility was calculated using Goldmann’s equation.
Results:
VEGF is secreted by both TM and SC cells in culture. In response to mechanical stretch, TM cells increased VEGF secretion by 44% (p<0.02). Similarly, VEGF levels were higher in response to dex at 3, 5, and 7 days (501+/-106 ng/ml VEGF vs 329+/-18 ng/ml control, p<0.05), and as a control, myocilin expression increased in dex-treated cells. Interestingly, SC monolayers showed no change in TEER in response to VEGF, while VEGF resulted in a 25% increase (p<0.04) in outflow facility in vivo.
Conclusions:
Our study demonstrates that cells of the conventional outflow tract secrete endogenous levels of VEGF that increase with stretch. Importantly, exogenous VEGF increases outflow facility in vivo, suggesting that anti-VEGF therapy may affect outflow regulation and IOP by altering endogenous VEGF tone.
Keywords: 748 vascular endothelial growth factor •
735 trabecular meshwork •
568 intraocular pressure