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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

Vasoinhibins block BK-induced reduction of transendothelial resistance and morphological changes in actin cytoskeleton. Time course of trans-electrical resistance (TER) in MRCEC (A) and MBCEC (B) monolayers cultured in complete medium (Ctl) with or without 10 μM BK and 10 nM vasoinhibins (Vi). (C) TER in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK and 10 nM Vi. (D) Expanded early time values of experiment (C). In (A–D), values are mean ± s.e.m. from 3 independent experiments normalized to the control; *P < 0.05 vs. Ctl. MBCEC and MRCEC were cultured on inserts with pore sizes of 0.4 μm while BUVEC cells were cultured on inserts with pore sizes of 8.0 μm. (E) BUVEC were cultured in complete medium (Ctl) with or without 10 μM BK and 10 nM Vi 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 2: Vasoinhibins block BK-induced reduction of transendothelial resistance and morphological changes in actin cytoskeleton. Time course of trans-electrical resistance (TER) in MRCEC (A) and MBCEC (B) monolayers cultured in complete medium (Ctl) with or without 10 μM BK and 10 nM vasoinhibins (Vi). (C) TER in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK and 10 nM Vi. (D) Expanded early time values of experiment (C). In (A–D), values are mean ± s.e.m. from 3 independent experiments normalized to the control; *P < 0.05 vs. Ctl. MBCEC and MRCEC were cultured on inserts with pore sizes of 0.4 μm while BUVEC cells were cultured on inserts with pore sizes of 8.0 μm. (E) BUVEC were cultured in complete medium (Ctl) with or without 10 μM BK and 10 nM Vi for 15 min and then actin cytoskeleton (F-actin) distribution was determined using rhodamine-phalloidin. Representative fields are shown. Scale bar, 10 μm.

Mentions: The inner BRB refers to the layer of capillaries in the inner retina with very low permeability, due to the interactions between vascular endothelial cells and surrounding cells (pericytes and macroglia) (Klaassen et al., 2013). Freshly isolated mouse retinal (Figure 2A) and brain (Figure 2B) capillary endothelial cells showed stable resistance over time. BK induced a transient decrease in TER that was maximal at 15 min and was fully prevented by vasoinhibins (Figures 2A,B). Vasoinhibins alone had no effect (Figures 2A,B). Monolayers of BUVEC that express B2 receptors (Wohlfart et al., 1997), also showed stable resistance over time (Figure 2C). BK induced a transient decrease in TER that was maximal at 10 min (Figures 2C,D) and was fully prevented by vasoinhibins (Figure 2C). Vasoinhibins alone had no effect (Figure 2C). The cytoskeleton is composed of actin microfilaments, which are critical for endothelial cell permeability (Dudek and Garcia, 2001). Treatment with BK induced F-actin redistribution and stress fiber formation in BUVEC (Figure 2E). These effects were blocked by vasoinhibins, and vasoinhibins had no effect alone (Figure 2E).


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)

Vasoinhibins block BK-induced reduction of transendothelial resistance and morphological changes in actin cytoskeleton. Time course of trans-electrical resistance (TER) in MRCEC (A) and MBCEC (B) monolayers cultured in complete medium (Ctl) with or without 10 μM BK and 10 nM vasoinhibins (Vi). (C) TER in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK and 10 nM Vi. (D) Expanded early time values of experiment (C). In (A–D), values are mean ± s.e.m. from 3 independent experiments normalized to the control; *P < 0.05 vs. Ctl. MBCEC and MRCEC were cultured on inserts with pore sizes of 0.4 μm while BUVEC cells were cultured on inserts with pore sizes of 8.0 μm. (E) BUVEC were cultured in complete medium (Ctl) with or without 10 μM BK and 10 nM Vi 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

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

Figure 2: Vasoinhibins block BK-induced reduction of transendothelial resistance and morphological changes in actin cytoskeleton. Time course of trans-electrical resistance (TER) in MRCEC (A) and MBCEC (B) monolayers cultured in complete medium (Ctl) with or without 10 μM BK and 10 nM vasoinhibins (Vi). (C) TER in BUVEC monolayers cultured in complete medium (Ctl) with or without 10 μM BK and 10 nM Vi. (D) Expanded early time values of experiment (C). In (A–D), values are mean ± s.e.m. from 3 independent experiments normalized to the control; *P < 0.05 vs. Ctl. MBCEC and MRCEC were cultured on inserts with pore sizes of 0.4 μm while BUVEC cells were cultured on inserts with pore sizes of 8.0 μm. (E) BUVEC were cultured in complete medium (Ctl) with or without 10 μM BK and 10 nM Vi for 15 min and then actin cytoskeleton (F-actin) distribution was determined using rhodamine-phalloidin. Representative fields are shown. Scale bar, 10 μm.
Mentions: The inner BRB refers to the layer of capillaries in the inner retina with very low permeability, due to the interactions between vascular endothelial cells and surrounding cells (pericytes and macroglia) (Klaassen et al., 2013). Freshly isolated mouse retinal (Figure 2A) and brain (Figure 2B) capillary endothelial cells showed stable resistance over time. BK induced a transient decrease in TER that was maximal at 15 min and was fully prevented by vasoinhibins (Figures 2A,B). Vasoinhibins alone had no effect (Figures 2A,B). Monolayers of BUVEC that express B2 receptors (Wohlfart et al., 1997), also showed stable resistance over time (Figure 2C). BK induced a transient decrease in TER that was maximal at 10 min (Figures 2C,D) and was fully prevented by vasoinhibins (Figure 2C). Vasoinhibins alone had no effect (Figure 2C). The cytoskeleton is composed of actin microfilaments, which are critical for endothelial cell permeability (Dudek and Garcia, 2001). Treatment with BK induced F-actin redistribution and stress fiber formation in BUVEC (Figure 2E). These effects were blocked by vasoinhibins, and vasoinhibins had no effect alone (Figure 2E).

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