Vasoinhibins Prevent the Diabetic Retinopathy-Associated Vascular Leakage in vivo via Protein Phosphatase 2A-Dependent eNOS Inactivation

Abstract

Purpose: : Vasoinhibins are a family of peptides derived from prolactin that inhibit angiogenesis and vasodilation in the retina. Here, we evaluated whether vasoinhibins prevent increased retinal vascular permeability (RVP) in response to intravitreous injection of VEGF or of vitreous from patients with diabetic retinopathy (DR). We further analyzed whether this effect occurs via protein phosphatase 2A (PP2A)-dependent dephosphorylation/inactivation of endothelial nitric oxide synthase (eNOS).

Methods: : Wistar rats were injected intravitreally with VEGF (300 ng), or with 2 µl of human vitreous from patients with DR or non-diabetic corpse donors, with or without vasoinhibins (1µg). RVP was investigated by the Evans blue method. Retinal NOS activity and eNOS phosphorylation were measured by the [³H]L-citrulline assay and Western blots, respectively.

Results: : Vasoinhibins blocked RVP elevated in response to intravitreous injection of VEGF or of vitreous from patients with DR. The extent of inhibition by vasoinhibins was similar to that following immunodepletion of VEGF from DR vitreous or blockage of NO synthesis with L-NAME. Vasoinhibins prevented VEGF-induced eNOS phosphorylation at Ser¹¹⁷⁹, and NOS activation in the retina. PP2A leads to eNOS dephosphorylation at Ser¹¹⁷⁹ and the PP2A inhibitor okadaic acid blocked the effect of vasoinhibins on RVP.

Conclusions: : The present study shows that vasoinhibins prevent the DR-associated increase of RVP. The effect involves the inhibition of VEGF-induced eNOS activation due to dephosphorylation of eNOS by PP2A. Our findings suggest that vasoinhibins may be developed as protective agents against diabetic macular edema.

Keywords: diabetic retinopathy • nitric oxide • vascular endothelial growth factor