Protective effect of PACAP-38 on retinal pigmented epithelium in an in vitro and in vivo model of diabetic retinopathy through EGFR-dependent mechanism

Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes. In the last years, several in vivo studies have demonstrated the protective role of pituitary adenylate cyclase-activating peptide (PACAP-38) to counteract several alterations occurring during DR. Recently, different studies have demonstrated that some PACAP-38 effects are mediated by EGFR trans-activation, although no data exist regarding the link between this peptide and EGFR in DR. The aim of the present study has been to investigate whether retinal effect of PACAP-38 against high glucose damage is mediated by EGFR phosphorylation. Diabetes was induced by a single injection of streptozotocin (STZ) in rats. After 1 week, a group of animals was treated with a single intravitreal injection of 100 μM PACAP-38 or saline solution. Immunohistochemistry and western blot analysis have demonstrated that intravitreal injection of PACAP-38 induced p-EGFR over-expression in retina of diabetic rats. Several pathogenic mechanisms may contribute to diabetic retinopathy including BRB alteration. To better clarify the relationship between PACAP-38 and EGFR, we have also carried out a study on ARPE-19 cells, representing a model in vitro of outer BRB. Our results have shown that PACAP-38 treatment improved cell viability in ARPE-19 cells exposed to hyperglycemic/hypoxic insult mimicking tissue microenvironment occurring in DR. Binding to PAC1R, peptide induces EGFR phosphorylation via PKA-signaling cascade stimulation. EGFR trans-activation triggers MAPK/ERK signaling pathway involved in cell survival and proliferation. In conclusion, data have suggested that PACAP-38 acts through EGFR phosphorylation in DR and this effect particularly occurs on RPE layer.