Vasoinhibins prevent retinal vasopermeability associated with diabetic retinopathy in rats via protein phosphatase 2A–dependent eNOS inactivation
Celina García, … , Gonzalo Martínez de la Escalera, Carmen Clapp
Celina García, … , Gonzalo Martínez de la Escalera, Carmen Clapp
Published June 2, 2008; First published May 22, 2008
Citation Information: J Clin Invest. 2008;118(6):2291-2300. https://doi.org/10.1172/JCI34508.
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Research Article Ophthalmology

Vasoinhibins prevent retinal vasopermeability associated with diabetic retinopathy in rats via protein phosphatase 2A–dependent eNOS inactivation

Abstract

Increased retinal vasopermeability contributes to diabetic retinopathy, the leading cause of blindness in working-age adults. Despite clinical progress, effective therapy remains a major need. Vasoinhibins, a family of peptides derived from the protein hormone prolactin (and inclusive of the 16-kDa fragment of prolactin), antagonize the proangiogenic effects of VEGF, a primary mediator of retinal vasopermeability. Here, we demonstrate what we believe to be a novel function of vasoinhibins as inhibitors of the increased retinal vasopermeability associated with diabetic retinopathy. Vasoinhibins inhibited VEGF-induced vasopermeability in bovine aortic and rat retinal capillary endothelial cells in vitro. In vivo, vasoinhibins blocked retinal vasopermeability in diabetic rats and in response to intravitreous injection of VEGF or of vitreous from patients with diabetic retinopathy. Inhibition by vasoinhibins was similar to that achieved following immunodepletion of VEGF from human diabetic retinopathy vitreous or blockage of NO synthesis, suggesting that vasoinhibins inhibit VEGF-induced NOS activation. We further showed that vasoinhibins activate protein phosphatase 2A (PP2A), leading to eNOS dephosphorylation at Ser1179 and, thereby, eNOS inactivation. Moreover, intravitreous injection of okadaic acid, a PP2A inhibitor, blocked the vasoinhibin effect on endothelial cell permeability and retinal vasopermeability. These results suggest that vasoinhibins have the potential to be developed as new therapeutic agents to control the excessive retinal vasopermeability observed in diabetic retinopathy and other vasoproliferative retinopathies.

Authors

Celina García, Jorge Aranda, Edith Arnold, Stéphanie Thébault, Yazmín Macotela, Fernando López-Casillas, Valentín Mendoza, Hugo Quiroz-Mercado, Hebert Luis Hernández-Montiel, Sue-Hwa Lin, Gonzalo Martínez de la Escalera, Carmen Clapp

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Figure 1

Vasoinhibins (Vi) prevent VEGF-induced vasopermeability, eNOS activity, and eNOS phosphorylation in endothelial cells.

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Vasoinhibins (Vi) prevent VEGF-induced vasopermeability, eNOS activity, ...
(A) Line diagrams depicting the different protocols used in experiments represented in B, C, and D. (B) Vasopermeability was measured by HRP leakage in BAEC monolayers either untreated (ctl) or treated with 5 nM VEGF in the absence or presence of 0.1, 0.5, 1, 5, 20, or 50 nM vasoinhibins for 48 hours. BAECs were incubated with VEGF combined with 1 mM l-NAME. Values are mean ± SEM of 5 independent experiments. *P < 0.05 versus ctl; **P < 0.05 versus VEGF. (C) eNOS activity was determined by the [3H]l-citrulline assay in BAECs incubated or not with 5 nM VEGF, alone or combined with increasing concentrations of vasoinhibins for 1 hour. Values are mean ± SEM of 5 independent experiments. *P < 0.05 versus no treatment; **P < 0.05 versus VEGF. (D) Western blot analysis of eNOS-Ser1179 phosphorylation in BAECs preincubated or not with 20 nM vasoinhibins for 1 hour and then treated with 5 nM VEGF for 1, 5, or 10 minutes. Total eNOS served as loading control. (E) Quantification of eNOS phosphorylation by densitometry after normalization for total eNOS. Values are mean ± SEM of 3 independent experiments. *P < 0.05 versus no treatment; **P < 0.05 versus VEGF.