Ameliorative effect of PACAP and VIP against increased permeability in a model of outer blood retinal barrier

Diabetic retinopathy (DR) is one of the leading causes of blindness and vision lossin adults. A recent study has demonstrated that compromised tight junctions, basementmembrane thickening and increased vascular permeability contribute to the disruptionof the outer blood-retinal barrier (BRB) in DR. The outer BRB is formed byretinal pigmented epithelial cells and controls the flow of solutes and fluid from thechoroidal vasculature to the outer retina. Previous studies have shown that pituitaryadenylate cyclase activating polypeptide (PACAP) and the related peptide vasoactiveintestinal polypeptide (VIP) are protective against several types of retinal injuries,including DR. However, whether these peptides are involved in maintenance ofouter BRB function during DR remains to be elucidated. Here, using an in vitro modelof dysfunctional outer BRB, we explored the effects of both PACAP and VIP. Retinalpigment epithelial cells (ARPE19) were cultured for 18 days either in normal glucose(NG, 5.5 mM) or in high glucose (HG, 25mM). In addition, to mimic the inflammatoryaspect of the diabetic milieu, HG cells were also treated with IL-1β (HG + IL-1β).Effects of PACAP or VIP on cells permeability were evaluated by measuring bothtransepithelial electrical resistance (TEER) and apical-to-basolateral movements of fluoresceinisothyocyanate (FITC) dextran. mRNA and protein expression of tight junction-related proteins (ZO-1, occludin and claudin, respectively) were evaluated byreal-time PCR (RT-PCR) and Western blot analyses. Our results show that cells grownin HG or in HG + IL-1β show significantly increased FITC-dextran diffusion, which isparalleled by a decrease in TEER as compared to NG cultures. Treatment with eitherPACAP or VIP were able to reverse both of these effects. Induction of claudin expressionwas observed both in cell cultures grown in HG alone or cotreated with IL-1β,which was totally abrogated by PACAP and, to a minor extent, by VIP. Neither ZO-1nor occludin seemed to be affected in our experimental model. In conclusion, the presentfinding show that PACAP and VIP are able to counteract HG- or HG + IL-1β-induced damage in ARPE19 cells, suggesting that both peptides might be relevant tothe maintenance of outer BRB function during DR, possibly through the modulationof claudin.