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 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 5

Vasoinhibins prevent VEGF-induced retinal vasopermeability in vivo via NOS inactivation.

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Vasoinhibins prevent VEGF-induced retinal vasopermeability in vivo via N...
(A) Retinal vasopermeability was determined by Evans blue assay of retinal extracts from rats injected intravitreally 24 hours earlier with PBS (ctl), 1 μM vasoinhibins; 2 μM OA; 260 nM VEGF; VEGF and vasoinhibins; VEGF and OA; or VEGF, vasoinhibins, and OA, as indicated. l-NAME (1.8 mM) was administered in the drinking water for 15 days, and retinas were analyzed for retinal vasopermeability. Values are mean ± SEM of 10 independent samples. *P < 0.05 versus control; **P < 0.05 versus VEGF; #P < 0.05 versus vasoinhibins and VEGF. (B) The [3H]l-citrulline assay was used to determine NOS activity in retinal extracts from rats injected intravitreally 24 hours earlier with PBS, 1 μM vasoinhibins, 260 nM VEGF, or VEGF plus vasoinhibins. Values are mean ± SEM of 10 independent samples. *P < 0.05 versus control; **P < 0.05 versus VEGF. VEGF and vasoinhibin concentrations refer to the final, intravitreal levels. (C) Vasopermeability was measured by HRP leakage in RRCECs untreated (ctl), treated with either 5 nM VEGF or VEGF combined with 1 mM l-NAME or 0.1, 0.5, 1, 5, 20, or 50 nM vasoinhibins for 48 hours as indicated. Values are mean ± SEM of 6 independent experiments. *P < 0.05 versus untreated control; **P < 0.05 versus VEGF. (D) eNOS activity was determined in RRCECs treated with or without 5 nM VEGF alone or together with 20 nM vasoinhibins, in the presence or absence of 50 nM OA for 1 hour. Values are mean ± SEM of 3 independent experiments. *P < 0.05 versus no treatment; **P < 0.05 versus VEGF; #P < 0.05 versus VEGF and vasoinhibins. (E) Western blot analysis of eNOS-Ser1179 phosphorylation in rat retinas 3 hours after injection with PBS (ctl), VEGF (260 nM), VEGF plus vasoinhibins (1 μM), or vasoinhibins alone. Total eNOS served as loading control. (F) Quantification of eNOS phosphorylation by densitometry normalized to total eNOS. Values are mean ± SEM of 3 independent experiments. *P < 0.05 versus control; **P < 0.05 versus VEGF.