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Vasoinhibins regulate the inner and outer blood-retinal barrier and limit retinal oxidative stress.

Arredondo Zamarripa D, Díaz-Lezama N, Meléndez García R, Chávez Balderas J, Adán N, Ledesma-Colunga MG, Arnold E, Clapp C, Thebault S - Front Cell Neurosci (2014)

Bottom Line: BK transiently decreased human RPE (ARPE-19) cell monolayer resistance, and this effect was blocked by vasoinhibins, L-NAME, and NAC.DETANONOate reverted the blocking effect of vasoinhibins.These effects on RPE resistance coincided with actin cytoskeleton redistribution.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México Querétaro, México.

ABSTRACT
Vasoinhibins are prolactin fragments present in the retina, where they have been shown to prevent the hypervasopermeability associated with diabetes. Enhanced bradykinin (BK) production contributes to the increased transport through the blood-retina barrier (BRB) in diabetes. Here, we studied if vasoinhibins regulate BRB permeability by targeting the vascular endothelium and retinal pigment epithelium (RPE) components of this barrier. Intravitreal injection of BK in male rats increased BRB permeability. Vasoinhibins prevented this effect, as did the B2 receptor antagonist Hoe-140. BK induced a transient decrease in mouse retinal and brain capillary endothelial monolayer resistance that was blocked by vasoinhibins. Both vasoinhibins and the nitric oxide (NO) synthase inhibitor L-NAME, but not the antioxidant N-acetyl cysteine (NAC), blocked the transient decrease in bovine umbilical vein endothelial cell (BUVEC) monolayer resistance induced by BK; this block was reversed by the NO donor DETANONOate. Vasoinhibins also prevented the BK-induced actin cytoskeleton redistribution, as did L-NAME. BK transiently decreased human RPE (ARPE-19) cell monolayer resistance, and this effect was blocked by vasoinhibins, L-NAME, and NAC. DETANONOate reverted the blocking effect of vasoinhibins. Similar to BK, the radical initiator Luperox induced a reduction in ARPE-19 cell monolayer resistance, which was prevented by vasoinhibins. These effects on RPE resistance coincided with actin cytoskeleton redistribution. Intravitreal injection of vasoinhibins reduced the levels of reactive oxygen species (ROS) in retinas of streptozotocin-induced diabetic rats, particularly in the RPE and capillary-containing layers. Thus, vasoinhibins reduce BRB permeability by targeting both its main inner and outer components through NO- and ROS-dependent pathways, offering potential treatment strategies against diabetic retinopathies.

No MeSH data available.


Related in: MedlinePlus

ROS do not participate in the vasoinhibin-mediated inhibition of transendothelial resistance reduction and actin cytoskeleton rearrangement induced by BK. (A,C) Time course of trans-electrical resistance (TER) in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK and the antioxidant N-acetyl cysteine (NAC, 10 mM) or with or without 500 μM Luperox and vasoinhibins (Vi, 10 nM). BUVEC were cultured on inserts with pore sizes of 8.0 μm. (B) Corresponding quantification of TER values, 10 min after treatment initiation. Values correspond to the mean ± s.e.m. from 3 independent experiments. *P < 0.05. NS, not significant. (D) BUVEC were cultured in complete medium (Ctl) with or without 10 μM BK and 10 mM NAC or with or without 500 μM Luperox and Vi (10 nM) for 15 min, and then actin cytoskeleton (F-actin) distribution was determined using rhodamine-phalloidin. Representative fields are shown. Scale bar, 10 μm. (E) Mitochondrial membrane potential changes using JC-1 dye in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK or 500 μM Luperox and 10 nM Vi. Values correspond to the mean ± s.e.m. from 3 independent experiments. *P < 0.05. NS, not significant. (F) Cytosolic levels of superoxide in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK. Values correspond to the mean ± s.e.m. of 8 repeats per condition from 3 independent experiments.
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Figure 4: ROS do not participate in the vasoinhibin-mediated inhibition of transendothelial resistance reduction and actin cytoskeleton rearrangement induced by BK. (A,C) Time course of trans-electrical resistance (TER) in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK and the antioxidant N-acetyl cysteine (NAC, 10 mM) or with or without 500 μM Luperox and vasoinhibins (Vi, 10 nM). BUVEC were cultured on inserts with pore sizes of 8.0 μm. (B) Corresponding quantification of TER values, 10 min after treatment initiation. Values correspond to the mean ± s.e.m. from 3 independent experiments. *P < 0.05. NS, not significant. (D) BUVEC were cultured in complete medium (Ctl) with or without 10 μM BK and 10 mM NAC or with or without 500 μM Luperox and Vi (10 nM) for 15 min, and then actin cytoskeleton (F-actin) distribution was determined using rhodamine-phalloidin. Representative fields are shown. Scale bar, 10 μm. (E) Mitochondrial membrane potential changes using JC-1 dye in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK or 500 μM Luperox and 10 nM Vi. Values correspond to the mean ± s.e.m. from 3 independent experiments. *P < 0.05. NS, not significant. (F) Cytosolic levels of superoxide in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK. Values correspond to the mean ± s.e.m. of 8 repeats per condition from 3 independent experiments.

Mentions: Evidence suggests that increased retinal vascular permeability in response to agonists such as BK is associated with increased ROS load (Wohlfart et al., 1997; Fong et al., 2010), which can be reduced inside the cell by free radical scavengers like NAC (Zavodnik et al., 2013). We observed that NAC did not block BK-induced reduction of TER in BUVEC (Figure 4A). Figure 4B shows the quantification of TER values at 15 min, when the BK effect was maximal. On the other hand, the free radical initiator Luperox (Zavodnik et al., 2013) did reduce TER in BUVEC after 30 min, an effect that persisted throughout the 90-min recording period (Figure 4C). Vasoinhibins delayed the action of Luperox by 15 min, without affecting the magnitude of its effect (Figure 4C). Furthermore, the BK-induced F-actin redistribution and stress fiber formation was not prevented by the antioxidant NAC, and NAC alone had no effect (Figure 4D). Luperox did not significantly affect F-actin distribution, nor did it when co-administered with vasoinhibins (Figure 4D). Further, we assessed the levels of ROS generated by mitochondrial oxidative phosphorylation, using the JC-1 probe. Levels of ROS were assessed in BUVEC monolayers treated with BK for 15 min, when its decrease of TER is maximal. BK did not modify intracellular ROS levels compared with untreated BUVEC monolayers (Figure 4E). A 15- and 45-min exposure of BUVEC to Luperox decreased the 530/590 optical density ratio, indicating increased levels of intracellular ROS (Figure 4E). Concomittant administration of vasoinhibins did not prevent the Luperox-induced production of ROS. While ROS levels were not modified after a 15-min incubation with vasoinhibins alone, they were reduced when the incubation with vasoinhibins was extended to 45 min (Figure 4E). In addition, ROS can be generated from oxidoreductase enzymes and metal-catalyzed oxidation. However, BK did not modify cytosolic levels of superoxide in BUVEC (Figure 4F). The observations that BK and Luperox acted with different kinetics, that NAC did not block the BK effect, and that BK does not modify the levels of ROS generated as byproducts during mitochondrial electron transport or as intermediates of metal-catalyzed oxidation reactions, indicate that ROS do not contribute to BK action. Our data also show that vasoinhibins delay the ROS effect on transendothelial resistance and that the vasoinhibins per se reduce the intraendothelial levels of ROS.


Vasoinhibins regulate the inner and outer blood-retinal barrier and limit retinal oxidative stress.

Arredondo Zamarripa D, Díaz-Lezama N, Meléndez García R, Chávez Balderas J, Adán N, Ledesma-Colunga MG, Arnold E, Clapp C, Thebault S - Front Cell Neurosci (2014)

ROS do not participate in the vasoinhibin-mediated inhibition of transendothelial resistance reduction and actin cytoskeleton rearrangement induced by BK. (A,C) Time course of trans-electrical resistance (TER) in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK and the antioxidant N-acetyl cysteine (NAC, 10 mM) or with or without 500 μM Luperox and vasoinhibins (Vi, 10 nM). BUVEC were cultured on inserts with pore sizes of 8.0 μm. (B) Corresponding quantification of TER values, 10 min after treatment initiation. Values correspond to the mean ± s.e.m. from 3 independent experiments. *P < 0.05. NS, not significant. (D) BUVEC were cultured in complete medium (Ctl) with or without 10 μM BK and 10 mM NAC or with or without 500 μM Luperox and Vi (10 nM) for 15 min, and then actin cytoskeleton (F-actin) distribution was determined using rhodamine-phalloidin. Representative fields are shown. Scale bar, 10 μm. (E) Mitochondrial membrane potential changes using JC-1 dye in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK or 500 μM Luperox and 10 nM Vi. Values correspond to the mean ± s.e.m. from 3 independent experiments. *P < 0.05. NS, not significant. (F) Cytosolic levels of superoxide in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK. Values correspond to the mean ± s.e.m. of 8 repeats per condition from 3 independent experiments.
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Figure 4: ROS do not participate in the vasoinhibin-mediated inhibition of transendothelial resistance reduction and actin cytoskeleton rearrangement induced by BK. (A,C) Time course of trans-electrical resistance (TER) in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK and the antioxidant N-acetyl cysteine (NAC, 10 mM) or with or without 500 μM Luperox and vasoinhibins (Vi, 10 nM). BUVEC were cultured on inserts with pore sizes of 8.0 μm. (B) Corresponding quantification of TER values, 10 min after treatment initiation. Values correspond to the mean ± s.e.m. from 3 independent experiments. *P < 0.05. NS, not significant. (D) BUVEC were cultured in complete medium (Ctl) with or without 10 μM BK and 10 mM NAC or with or without 500 μM Luperox and Vi (10 nM) for 15 min, and then actin cytoskeleton (F-actin) distribution was determined using rhodamine-phalloidin. Representative fields are shown. Scale bar, 10 μm. (E) Mitochondrial membrane potential changes using JC-1 dye in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK or 500 μM Luperox and 10 nM Vi. Values correspond to the mean ± s.e.m. from 3 independent experiments. *P < 0.05. NS, not significant. (F) Cytosolic levels of superoxide in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK. Values correspond to the mean ± s.e.m. of 8 repeats per condition from 3 independent experiments.
Mentions: Evidence suggests that increased retinal vascular permeability in response to agonists such as BK is associated with increased ROS load (Wohlfart et al., 1997; Fong et al., 2010), which can be reduced inside the cell by free radical scavengers like NAC (Zavodnik et al., 2013). We observed that NAC did not block BK-induced reduction of TER in BUVEC (Figure 4A). Figure 4B shows the quantification of TER values at 15 min, when the BK effect was maximal. On the other hand, the free radical initiator Luperox (Zavodnik et al., 2013) did reduce TER in BUVEC after 30 min, an effect that persisted throughout the 90-min recording period (Figure 4C). Vasoinhibins delayed the action of Luperox by 15 min, without affecting the magnitude of its effect (Figure 4C). Furthermore, the BK-induced F-actin redistribution and stress fiber formation was not prevented by the antioxidant NAC, and NAC alone had no effect (Figure 4D). Luperox did not significantly affect F-actin distribution, nor did it when co-administered with vasoinhibins (Figure 4D). Further, we assessed the levels of ROS generated by mitochondrial oxidative phosphorylation, using the JC-1 probe. Levels of ROS were assessed in BUVEC monolayers treated with BK for 15 min, when its decrease of TER is maximal. BK did not modify intracellular ROS levels compared with untreated BUVEC monolayers (Figure 4E). A 15- and 45-min exposure of BUVEC to Luperox decreased the 530/590 optical density ratio, indicating increased levels of intracellular ROS (Figure 4E). Concomittant administration of vasoinhibins did not prevent the Luperox-induced production of ROS. While ROS levels were not modified after a 15-min incubation with vasoinhibins alone, they were reduced when the incubation with vasoinhibins was extended to 45 min (Figure 4E). In addition, ROS can be generated from oxidoreductase enzymes and metal-catalyzed oxidation. However, BK did not modify cytosolic levels of superoxide in BUVEC (Figure 4F). The observations that BK and Luperox acted with different kinetics, that NAC did not block the BK effect, and that BK does not modify the levels of ROS generated as byproducts during mitochondrial electron transport or as intermediates of metal-catalyzed oxidation reactions, indicate that ROS do not contribute to BK action. Our data also show that vasoinhibins delay the ROS effect on transendothelial resistance and that the vasoinhibins per se reduce the intraendothelial levels of ROS.

Bottom Line: BK transiently decreased human RPE (ARPE-19) cell monolayer resistance, and this effect was blocked by vasoinhibins, L-NAME, and NAC.DETANONOate reverted the blocking effect of vasoinhibins.These effects on RPE resistance coincided with actin cytoskeleton redistribution.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México Querétaro, México.

ABSTRACT
Vasoinhibins are prolactin fragments present in the retina, where they have been shown to prevent the hypervasopermeability associated with diabetes. Enhanced bradykinin (BK) production contributes to the increased transport through the blood-retina barrier (BRB) in diabetes. Here, we studied if vasoinhibins regulate BRB permeability by targeting the vascular endothelium and retinal pigment epithelium (RPE) components of this barrier. Intravitreal injection of BK in male rats increased BRB permeability. Vasoinhibins prevented this effect, as did the B2 receptor antagonist Hoe-140. BK induced a transient decrease in mouse retinal and brain capillary endothelial monolayer resistance that was blocked by vasoinhibins. Both vasoinhibins and the nitric oxide (NO) synthase inhibitor L-NAME, but not the antioxidant N-acetyl cysteine (NAC), blocked the transient decrease in bovine umbilical vein endothelial cell (BUVEC) monolayer resistance induced by BK; this block was reversed by the NO donor DETANONOate. Vasoinhibins also prevented the BK-induced actin cytoskeleton redistribution, as did L-NAME. BK transiently decreased human RPE (ARPE-19) cell monolayer resistance, and this effect was blocked by vasoinhibins, L-NAME, and NAC. DETANONOate reverted the blocking effect of vasoinhibins. Similar to BK, the radical initiator Luperox induced a reduction in ARPE-19 cell monolayer resistance, which was prevented by vasoinhibins. These effects on RPE resistance coincided with actin cytoskeleton redistribution. Intravitreal injection of vasoinhibins reduced the levels of reactive oxygen species (ROS) in retinas of streptozotocin-induced diabetic rats, particularly in the RPE and capillary-containing layers. Thus, vasoinhibins reduce BRB permeability by targeting both its main inner and outer components through NO- and ROS-dependent pathways, offering potential treatment strategies against diabetic retinopathies.

No MeSH data available.


Related in: MedlinePlus