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Z. Ablonczy, K. Sambamurti, C. E. Crosson; Regulation of RPE Function by Juxtamembrane Proteases. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5346.
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Maintaining the integrity of the retinal pigment epithelium (RPE) is essential for normal visual acuity. The RPE is a primary source of vascular endothelial growth factor (VEGF) in the eye. Oxidative stress not only enhances VEGF secretion, but directs it toward the apical RPE surface, impairing the barrier function of the tissue. The actions of VEGF are antagonized by pigment-epithelium-derived factor (PEDF) through the activation of juxtamembrane proteases to process the VEGF receptors. Our objective is to understand how the PEDF/juxtamembrane protease system contributes to the development and resolution of retinal edema.
Monolayer cultures of fetal human RPE (fhRPE) cells were treated with apical VEGF-E and PEDF in the absence or presence of protease inhibitors. Trans-epithelial electrical resistance (TER) measurements assessed the barrier function. Protease actions were determined from immunoblots against the VEGF receptors. Fluid leakage and transport across the RPE of Dutch-belted rabbits was measured by fluorescein angiography and optical coherence tomography (OCT).
Apical VEGF-E induced a significant drop (from 1033 Ωcm2 to 647 Ωcm2) in the TER of fhRPE cell monolayers by 5 hours post-administration. The VEGF-E response was inhibited by PEDF, and the inhibition reversed by γ-secretase inhibitors (DAPT and LY411575). However, in contrast to VEGF-E treatment, recovery of resistance had not begun 5 hours after DAPT co-administration. The PEDF-induced inhibition of the VEGF-response was associated with VEGF-R2 ectodomain shedding and was blocked by GM6001 (an adamalysin inhibitor) and DAPT. Co-administration of VEGF and DAPT induced a significant increase in fluorescence emanating from the avascular area of the rabbit retina (RPE leakage) compared to VEGF treatment alone. Reabsorption of PBS-filled blebs was biphasic with an initial rate of 0.94 V0/h, and final rate of 0.06 V0/h. VEGF-filled blebs reabsorbed slowly, at a rate of 0.03 V0/h).
The data provide evidence that in RPE cultures with high barrier function, the anti-VEGF action of PEDF is mediated by juxtamembrane proteolysis of VEGF-R2. The released VEGF-R2 ectodomain is likely to act as a natural VEGF-trap molecule. The fluorescein angiography and high-accuracy fluid reabsorption measurements reinforce the significant role of the PEDF/juxtamembrane protease system in RPE function in vivo. Therefore, we hypothesize that when the juxtamembrane proteolytic activity declines (i.e., diabetes, or aging), VEGF-R2 activity is enhanced, disrupting RPE function and leading to the development of retinal edema.
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