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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 contribute to the inhibition of BK-induced decrease of ARPE-19 resistance and actin cytoskeleton rearrangement by vasoinhibins. (A,B) Time course of trans-electrical resistance (TER) in ARPE-19 monolayers cultured in complete medium (Ctl) with or without 10 μM BK and N-acetyl cysteine (NAC, 10 mM) or with or without 500 μM Luperox and vasoinhibins (Vi, 10 nM). Values correspond to the mean ± s.e.m. from 3 independent experiments. *P < 0.05 vs. Ctl. ARPE-19 cells were cultured on inserts with pore sizes of 0.4 μm. (C) ARPE-19 cells were cultured in complete medium (Ctl) with or without 10 μM BK and NAC (10 mM) 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. (D) Mitochondrial membrane potential changes using JC-1 dye in ARPE-19 monolayers cultured in complete medium (Ctl) with or without 10 μM BK or 500 μM Luperox and Vi (10 nM). Values correspond to the mean ± s.e.m. from 3 independent experiments. (E) Cytosolic levels of superoxide in ARPE-19 monolayers cultured in complete medium (Ctl) with or without 10 μM BK and NAC (10 mM) or with or without 10 μM BK and Vi (10 nM). Values correspond to the mean ± s.e.m. of 4–15 repeats per condition from 3 independent experiments. *P < 0.05 vs. Ctl. NS, not significant.
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Figure 6: ROS contribute to the inhibition of BK-induced decrease of ARPE-19 resistance and actin cytoskeleton rearrangement by vasoinhibins. (A,B) Time course of trans-electrical resistance (TER) in ARPE-19 monolayers cultured in complete medium (Ctl) with or without 10 μM BK and N-acetyl cysteine (NAC, 10 mM) or with or without 500 μM Luperox and vasoinhibins (Vi, 10 nM). Values correspond to the mean ± s.e.m. from 3 independent experiments. *P < 0.05 vs. Ctl. ARPE-19 cells were cultured on inserts with pore sizes of 0.4 μm. (C) ARPE-19 cells were cultured in complete medium (Ctl) with or without 10 μM BK and NAC (10 mM) 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. (D) Mitochondrial membrane potential changes using JC-1 dye in ARPE-19 monolayers cultured in complete medium (Ctl) with or without 10 μM BK or 500 μM Luperox and Vi (10 nM). Values correspond to the mean ± s.e.m. from 3 independent experiments. (E) Cytosolic levels of superoxide in ARPE-19 monolayers cultured in complete medium (Ctl) with or without 10 μM BK and NAC (10 mM) or with or without 10 μM BK and Vi (10 nM). Values correspond to the mean ± s.e.m. of 4–15 repeats per condition from 3 independent experiments. *P < 0.05 vs. Ctl. NS, not significant.

Mentions: The antioxidant NAC blocked BK-induced reduction of TER in ARPE-19 (Figure 6A), but alone, NAC had no effect. Also, Luperox induced a transient decrease in TER that was prevented by vasoinhibins (Figure 6B). Both the magnitude (15 ± 1 vs. 9 ± 2%) and kinetics (maximum at 10 vs. 15 min) of the Luperox effect were similar to those of BK. NAC had no effect alone but blocked the BK-induced F-actin redistribution (Figure 6C). Similarly to BK, Luperox induced F-actin redistribution, an effect that was inhibited by vasoinhibins (Figure 6C). Intracellular levels of ROS were assessed in the presence and absence of vasoinhibins with BK or Luperox for 15 min, when their effects on TER were maximal. BK did not modify ROS levels in ARPE-19, while Luperox increased them (Figure 6D). Alone or combined with BK or Luperox, vasoinhibins had no effect (Figure 6D). In contrast, BK increased cytosolic levels of superoxide in ARPE-19 at 15 min, and both NAC and vasoinhibins blocked this increase (Figure 6E).


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 contribute to the inhibition of BK-induced decrease of ARPE-19 resistance and actin cytoskeleton rearrangement by vasoinhibins. (A,B) Time course of trans-electrical resistance (TER) in ARPE-19 monolayers cultured in complete medium (Ctl) with or without 10 μM BK and N-acetyl cysteine (NAC, 10 mM) or with or without 500 μM Luperox and vasoinhibins (Vi, 10 nM). Values correspond to the mean ± s.e.m. from 3 independent experiments. *P < 0.05 vs. Ctl. ARPE-19 cells were cultured on inserts with pore sizes of 0.4 μm. (C) ARPE-19 cells were cultured in complete medium (Ctl) with or without 10 μM BK and NAC (10 mM) 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. (D) Mitochondrial membrane potential changes using JC-1 dye in ARPE-19 monolayers cultured in complete medium (Ctl) with or without 10 μM BK or 500 μM Luperox and Vi (10 nM). Values correspond to the mean ± s.e.m. from 3 independent experiments. (E) Cytosolic levels of superoxide in ARPE-19 monolayers cultured in complete medium (Ctl) with or without 10 μM BK and NAC (10 mM) or with or without 10 μM BK and Vi (10 nM). Values correspond to the mean ± s.e.m. of 4–15 repeats per condition from 3 independent experiments. *P < 0.05 vs. Ctl. NS, not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: ROS contribute to the inhibition of BK-induced decrease of ARPE-19 resistance and actin cytoskeleton rearrangement by vasoinhibins. (A,B) Time course of trans-electrical resistance (TER) in ARPE-19 monolayers cultured in complete medium (Ctl) with or without 10 μM BK and N-acetyl cysteine (NAC, 10 mM) or with or without 500 μM Luperox and vasoinhibins (Vi, 10 nM). Values correspond to the mean ± s.e.m. from 3 independent experiments. *P < 0.05 vs. Ctl. ARPE-19 cells were cultured on inserts with pore sizes of 0.4 μm. (C) ARPE-19 cells were cultured in complete medium (Ctl) with or without 10 μM BK and NAC (10 mM) 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. (D) Mitochondrial membrane potential changes using JC-1 dye in ARPE-19 monolayers cultured in complete medium (Ctl) with or without 10 μM BK or 500 μM Luperox and Vi (10 nM). Values correspond to the mean ± s.e.m. from 3 independent experiments. (E) Cytosolic levels of superoxide in ARPE-19 monolayers cultured in complete medium (Ctl) with or without 10 μM BK and NAC (10 mM) or with or without 10 μM BK and Vi (10 nM). Values correspond to the mean ± s.e.m. of 4–15 repeats per condition from 3 independent experiments. *P < 0.05 vs. Ctl. NS, not significant.
Mentions: The antioxidant NAC blocked BK-induced reduction of TER in ARPE-19 (Figure 6A), but alone, NAC had no effect. Also, Luperox induced a transient decrease in TER that was prevented by vasoinhibins (Figure 6B). Both the magnitude (15 ± 1 vs. 9 ± 2%) and kinetics (maximum at 10 vs. 15 min) of the Luperox effect were similar to those of BK. NAC had no effect alone but blocked the BK-induced F-actin redistribution (Figure 6C). Similarly to BK, Luperox induced F-actin redistribution, an effect that was inhibited by vasoinhibins (Figure 6C). Intracellular levels of ROS were assessed in the presence and absence of vasoinhibins with BK or Luperox for 15 min, when their effects on TER were maximal. BK did not modify ROS levels in ARPE-19, while Luperox increased them (Figure 6D). Alone or combined with BK or Luperox, vasoinhibins had no effect (Figure 6D). In contrast, BK increased cytosolic levels of superoxide in ARPE-19 at 15 min, and both NAC and vasoinhibins blocked this increase (Figure 6E).

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