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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

The inhibitory effect of vasoinhibins on BK-induced reduction of transendothelial resistance and actin cytoskeleton rearrangement depends on NO. (A) Time course of TER in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK and the NO synthase inhibitor L-NAME (10 mM). Values are mean ± s.e.m. from 3 independent experiments normalized to the control. *P < 0.05 vs. Ctl. (B) Rats were treated with L-NAME (1.8 mM) administered in the drinking water for 15 days, then their retinas were intravitreously injected with PBS (Ctl) or BK (1 nM), and evaluated 2 h later by the Evans blue dye assay. Values are mean ± s.e.m. normalized to control. *P < 0.05 from 8 to 16 independent observations. (C) Quantification of peak TER values (15 min after treatment start) in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK and 10 nM Vi in the absence (white bars) or presence (black bars) of the NO donor DETANONOate (10 μM). *P < 0.05 from 3 independent experiments. NS, not significant. BUVEC were cultured on inserts with pore sizes of 8.0 μm. (D) BUVEC were cultured in complete medium (Ctl) with or without 10 μM BK and 10 mM L-NAME, and with the NO donor DETANONOate (10 μM) in the presence and in the absence of BK 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.
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Figure 3: The inhibitory effect of vasoinhibins on BK-induced reduction of transendothelial resistance and actin cytoskeleton rearrangement depends on NO. (A) Time course of TER in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK and the NO synthase inhibitor L-NAME (10 mM). Values are mean ± s.e.m. from 3 independent experiments normalized to the control. *P < 0.05 vs. Ctl. (B) Rats were treated with L-NAME (1.8 mM) administered in the drinking water for 15 days, then their retinas were intravitreously injected with PBS (Ctl) or BK (1 nM), and evaluated 2 h later by the Evans blue dye assay. Values are mean ± s.e.m. normalized to control. *P < 0.05 from 8 to 16 independent observations. (C) Quantification of peak TER values (15 min after treatment start) in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK and 10 nM Vi in the absence (white bars) or presence (black bars) of the NO donor DETANONOate (10 μM). *P < 0.05 from 3 independent experiments. NS, not significant. BUVEC were cultured on inserts with pore sizes of 8.0 μm. (D) BUVEC were cultured in complete medium (Ctl) with or without 10 μM BK and 10 mM L-NAME, and with the NO donor DETANONOate (10 μM) in the presence and in the absence of BK 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.

Mentions: We observed that coadministration of the NOS inhibitor L-NAME (Rees et al., 1989) eliminated the BK-induced decrease in TER, but L-NAME alone had no effect (Figure 3A). Administration of L-NAME (1.8 mM) in the rats' drinking water for 15 days also counteracted BK action on BRB permeability (Figure 3B), mimicking the vasoinhibin effect (Figure 1A). In support of NO being essential for BK effects, the NO donor DETANONOate mimicked the BK effect on TER, and coadministration of DETANONOate did not enhance BK action (Figure 3C). We then asked whether the NO donor DETANONOate reverts the action of vasoinhibins in the presence of BK. Exogenous NO prevented vasoinhibin-mediated inhibition of BK-induced reduction of TER in BUVEC (Figure 3C). In addition, L-NAME prevented BK-induced redistribution of F-actin and stress fiber formation, but had no effect alone (Figure 3D). Also, DETANONOate induced F-actin redistribution and stress fiber formation similarly to BK, and coadministration of DETANONOate did not enhance BK action (Figure 3D). In this context, vasoinhibins were not able to block DETANONOate-induced F-actin rearrangement, and exogenous NO prevented vasoinhibin-mediated inhibition of BK-induced F-actin redistribution in BUVEC (Figure 3D). All together, these data show that NO mediates the BK-induced increase in endothelial cell monolayer permeability and actin cytoskeleton redistribution, and that vasoinhibins block these actions by acting upstream of NO production, as previously reported (Gonzalez et al., 2004; Thebault, 2011).


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)

The inhibitory effect of vasoinhibins on BK-induced reduction of transendothelial resistance and actin cytoskeleton rearrangement depends on NO. (A) Time course of TER in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK and the NO synthase inhibitor L-NAME (10 mM). Values are mean ± s.e.m. from 3 independent experiments normalized to the control. *P < 0.05 vs. Ctl. (B) Rats were treated with L-NAME (1.8 mM) administered in the drinking water for 15 days, then their retinas were intravitreously injected with PBS (Ctl) or BK (1 nM), and evaluated 2 h later by the Evans blue dye assay. Values are mean ± s.e.m. normalized to control. *P < 0.05 from 8 to 16 independent observations. (C) Quantification of peak TER values (15 min after treatment start) in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK and 10 nM Vi in the absence (white bars) or presence (black bars) of the NO donor DETANONOate (10 μM). *P < 0.05 from 3 independent experiments. NS, not significant. BUVEC were cultured on inserts with pore sizes of 8.0 μm. (D) BUVEC were cultured in complete medium (Ctl) with or without 10 μM BK and 10 mM L-NAME, and with the NO donor DETANONOate (10 μM) in the presence and in the absence of BK 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4202700&req=5

Figure 3: The inhibitory effect of vasoinhibins on BK-induced reduction of transendothelial resistance and actin cytoskeleton rearrangement depends on NO. (A) Time course of TER in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK and the NO synthase inhibitor L-NAME (10 mM). Values are mean ± s.e.m. from 3 independent experiments normalized to the control. *P < 0.05 vs. Ctl. (B) Rats were treated with L-NAME (1.8 mM) administered in the drinking water for 15 days, then their retinas were intravitreously injected with PBS (Ctl) or BK (1 nM), and evaluated 2 h later by the Evans blue dye assay. Values are mean ± s.e.m. normalized to control. *P < 0.05 from 8 to 16 independent observations. (C) Quantification of peak TER values (15 min after treatment start) in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK and 10 nM Vi in the absence (white bars) or presence (black bars) of the NO donor DETANONOate (10 μM). *P < 0.05 from 3 independent experiments. NS, not significant. BUVEC were cultured on inserts with pore sizes of 8.0 μm. (D) BUVEC were cultured in complete medium (Ctl) with or without 10 μM BK and 10 mM L-NAME, and with the NO donor DETANONOate (10 μM) in the presence and in the absence of BK 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.
Mentions: We observed that coadministration of the NOS inhibitor L-NAME (Rees et al., 1989) eliminated the BK-induced decrease in TER, but L-NAME alone had no effect (Figure 3A). Administration of L-NAME (1.8 mM) in the rats' drinking water for 15 days also counteracted BK action on BRB permeability (Figure 3B), mimicking the vasoinhibin effect (Figure 1A). In support of NO being essential for BK effects, the NO donor DETANONOate mimicked the BK effect on TER, and coadministration of DETANONOate did not enhance BK action (Figure 3C). We then asked whether the NO donor DETANONOate reverts the action of vasoinhibins in the presence of BK. Exogenous NO prevented vasoinhibin-mediated inhibition of BK-induced reduction of TER in BUVEC (Figure 3C). In addition, L-NAME prevented BK-induced redistribution of F-actin and stress fiber formation, but had no effect alone (Figure 3D). Also, DETANONOate induced F-actin redistribution and stress fiber formation similarly to BK, and coadministration of DETANONOate did not enhance BK action (Figure 3D). In this context, vasoinhibins were not able to block DETANONOate-induced F-actin rearrangement, and exogenous NO prevented vasoinhibin-mediated inhibition of BK-induced F-actin redistribution in BUVEC (Figure 3D). All together, these data show that NO mediates the BK-induced increase in endothelial cell monolayer permeability and actin cytoskeleton redistribution, and that vasoinhibins block these actions by acting upstream of NO production, as previously reported (Gonzalez et al., 2004; Thebault, 2011).

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