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.

Figures

Figure 1. Vasoinhibins (Vi) prevent VEGF-induced vasopermeability, eNOS activity, and eNOS phosphorylation in endothelial cells.

(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.

Figure 2. Vasoinhibins enhance PP2A activity in BAECs.

(A) Line diagrams depicting the different protocols used in experiments represented in B, C, and D. PP2A-like activity was measured in BAECs after treatment with increasing doses of vasoinhibins for 1 hour (B) and after incubation with 20 nM vasoinhibins for 1 or 30 minutes or 1, 2, or 3 hours (C). (D) PP2A-like activity was measured in BAECs after 1 hour of incubation in the absence or presence of 20 nM vasoinhibins, 50 nM OA, the combination of vasoinhibins and OA, 20 nM rapamycin (Rap), or the combination of rapamycin and OA. Bars correspond to the mean ± SEM from 4 independent experiments. *P < 0.05 versus no treatment; **P < 0.05 versus vasoinhibins or rapamycin alone.

Figure 3. Vasoinhibins inhibit VEGF-induced eNOS phosphorylation and eNOS activity via PP2A activation.

(A) Line diagrams depicting the different protocols used in experiments represented in B, C, and D. (B) Western blot analysis of eNOS-Ser1179 phosphorylation in BAECs preincubated or not with 50 nM OA for 10 minutes, then with or without 20 nM vasoinhibins for 1 hour, followed or not by a treatment with 5 nM VEGF for 5 minutes as indicated. Total eNOS served as loading control. (C) eNOS activity was determined by the [3H]l-citrulline assay in BAECs incubated or not with 5 nM VEGF, alone or combined with 20 nM vasoinhibins in the presence or absence of 50 nM OA for 1 hour. Values are mean ± SEM of 5 independent experiments. *P < 0.05 versus no treatment; **P < 0.05 versus VEGF; #P < 0.05 versus VEGF and vasoinhibins. (D) Vasopermeability was measured by HRP leakage in BAEC monolayers untreated (ctl) or treated for 48 hours with 50 nM OA, 5 nM VEGF, or the combination of VEGF and 20 nM vasoinhibins or VEGF, vasoinhibins, and OA. 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.

Figure 4. Vasoinhibins prevent VEGF-induced retinal hemorrhages and vasodilation in vivo.

(A) Representative flat mounts after the peroxidase reaction of a portion of the retina obtained from rats 24 hours after an intravitreous injection of PBS (ctl), 260 nM VEGF, or VEGF and 1 μM vasoinhibins. Each retina was digitized with a ×20 objective and tiled to present the entire preparation. Arrows indicate hemorrhage areas. Original magnification, ×6. (B) Magnification of areas indicated by rectangles (×7; ×40 objective) in the flat mounts shown in A to illustrate the vasodilation of the microcirculation. Insets in B were magnified by an additional 2-fold. Arrows indicate the width of blood vessels. Quantification of hemorrhage areas (C) and averaged diameter of blood vessels (D) from retina flat mounts from rats injected intravitreally 24 hours earlier with PBS, 260 nM VEGF, or VEGF and 1 μM Vi. VEGF and vasoinhibin concentrations refer to the final, intravitreal levels. Values are mean ± SEM of 6 independent samples. *P < 0.05 versus control; **P < 0.05 versus VEGF.

Figure 5. Vasoinhibins prevent VEGF-induced retinal vasopermeability in vivo via NOS inactivation.

(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.

Figure 6. Vasoinhibins prevent retinal vasopermeability induced by human diabetic vitreous.

(A) Evans blue evaluation of retinal vasopermeability in retinal extracts from rats injected intravitreally with PBS (ctl, n = 5), nondiabetic vitreous (2 μl, n = 5), nondiabetic vitreous plus vasoinhibins (1 μM, n = 5), nondiabetic vitreous depleted of VEGF (anti-VEGF, 2 μl, n = 3), diabetic vitreous (2 μl, n = 6), diabetic vitreous plus vasoinhibins (1 μM, n = 6), or diabetic vitreous depleted of VEGF (anti-VEGF, 2 μl, n = 3). The same VEGF-depleted vitreous was used for the experiments in A and B. l-NAME (1.8 mM) was administered in drinking water for 15 days, and rat retinas that had been intravitreally injected with diabetic vitreous were subsequently analyzed for retinal vasopermeability. Values are mean ± SEM of n independent experiments. *P < 0.05 versus ctl; #P < 0.05 versus diabetic vitreous. (B) Analysis of VEGF concentration (pg/ml) by ELISA in nondiabetic vitreous, nondiabetic vitreous depleted of VEGF (anti-VEGF), diabetic vitreous, and diabetic vitreous depleted of VEGF (anti-VEGF). The VEGF content of the vitreous samples was reduced by immunoprecipitation with anti-VEGF antibody. Values are mean ± SEM of 3 independent vitreous samples. *P < 0.05 versus nondiabetic vitreous; **P < 0.05 versus diabetic vitreous. (C) Evans blue evaluation of retinal vasopermeability in retinal extracts from nondiabetic rats and diabetic rats (induced by streptozotocin), injected with PBS or vasoinhibins (1 μM). Values are mean ± SEM of 6 independent retinal extracts. *P < 0.05 versus PBS-injected nondiabetic rats; **P < 0.05 versus PBS-injected diabetic rats. Vasoinhibin concentrations refer to the final, intravitreal values.