Dickkopf Protein 3 Inhibits VEGF Signaling and Bioactivity in Bovine Retina Endothelial Cells

Abstract

Purpose:: Using proteomic analyses, we found a reduction of the Wnt signaling antagonist Dickkopf protein 3 (DKK3) in the vitreous of patients with PDR. DKK3 has tumor suppressor, apoptosis and anti-proliferative activities and is involved in cellular migration and differentiation. We have shown that DKK3 is expressed by retinal pericytes and endothelial cells (REC) and that its expression is decreased in diabetic retina and by high glucose concentrations in culture. We now report that DKK3 inhibits VEGF-induced signaling, suppressing VEGF-induced REC proliferation, REC migration and retinal vascular permeability (RVP) in rats.

Methods:: Primary cultures of freshly isolated bovine REC were assayed for bromodeoxyridine (BrdU) incorporation. Cell migration from monlayer injury was quantitated. VEGF-induced VEGF receptor 2 (VEGF-R2) and Erk phosphorylation were evaluated with western blotting and immunoprecipitation. VEGF-induced rat RVP was quantified by evans blue permeation technique.

Results:: Recombinant human DKK3 (rhDKK3) inhibited VEGF-induced REC BrdU incorporation in dose dependent manner with 100% inhibition observed at 10µg/ml. rhDKK3 inhibited both serum- (30%) and VEGF- induced (70%) REC migration in a dose dependent manner. rhDKK3 inhibited VEGF-induced Erk phosphorylation by 50% at 10 µg/ml (p=0.012) and partially inhibited VEGF-induced VEGF-R2 phosphorylation. In rats, 2.5µg/eye rhDKK3 inhibited VEGF-induced RVP by 50% (p=0.013).

Conclusions:: rhDKK3 inhibits VEGF-induced REC proliferation, migration, VEGF-R2 and Erk phosphorylation as well as VEGF-induced retinal vascular permeability in vivo. Since DKK3 is reduced in the vitreous of patients with PDR, these findings suggest that DKK3 may have a regulatory role in mediating advanced diabetic retinal complications.

Keywords: diabetic retinopathy • vascular cells • proliferation