Atrial Natriuretic Peptide Prevents Disruption of RPE Barrier Function by VEGF

Abstract

Purpose: : Wet AMD is characterized by appearance of subretinal fluid. Normally, the fluid accumulation is prevented by the retinal pigment epithelium (RPE), which maintains the extracellular environment in the outer retina. Vascular endothelial growth factor (VEGF) plays a critical role in the breakdown of RPE barrier function and it is hypothesized that anti-permeability agents can counteract VEGF action. Atrial natriuretic peptide (ANP) is expressed in the normal eye and its receptors are found in the RPE. It has recently been recognized that ANP has anti-permeability properties; however, the impact of ANP on RPE function is unknown.

Methods: : Trans-epithelial electrical resistance (TEER) measurements were utilized to assess the barrier function of monolayer-cultured RPE cells in Transwell filters. The cells were treated with 1 pg/mL to 100 ng/mL apical VEGF or VEGF-E in the absence or presence of 1 pM to 1 mM apical or basolateral ANP. TEER was monitored for up to 5 hours post drug-administration. Involvement of of natriuretic peptide receptors (NPR) was confirmed by pretreatment with the NPR antagonist isatin (100 µM).

Results: : VEGF and VEGF-E induce a rapid breakdown of RPE barrier function. TEER of RPE cell monolayers decreases 35% within 2 hours and 45% within 5 hours after VEGF-R2 agonist treatments. Apically-administered ANP prevented the barrier breakdown induced by VEGF or VEGF-E in a concentration-dependent manner, with an IC₅₀ of 1 nM. The inhibitory actions of ANP were sustained for up to 5 hours when co-administered with VEGF-R2 agonists. The ANP-induced effects were blocked by pretreatment with 100 µM isatin. Basolateral ANP administration had no significant impact on the VEGF-induced decrease in RPE permeability.

Conclusions: : Our data provide novel evidence of an important role for ANP in countering VEGF action on RPE barrier function. ANP effectively reverses the impact of VEGF through the NPR receptors. Similar to VEGF, RPE’s response to ANP is polarized, as only apical administration caused significant inhibitory actions. The role of ANP in vivo and the mechanisms underlying its inhibitory effects are currently under investigation.

Keywords: retinal pigment epithelium • edema • vascular endothelial growth factor