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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 prevent BK-induced increase in BRB permeability similarly to a kinin B2 receptor antagonist. (A) Evaluation of the Evans blue dye content in retinas of rats intravitreously injected 4 h earlier with PBS (Ctl), BK (1 nM), BK combined with either vasoinhibins (Vi, 1 μM) or Hoe-140 (Hoe, 3 μM), Vi alone, or Hoe alone. Values are mean ± s.e.m. normalized to the control (n = 8–16 per group; *P < 0.05). (B–D) Retinas of rats that were intravitreously injected 4 h earlier with PBS, BK, Vi, or BK combined with Vi were analyzed for kinin B2 receptor mRNA (B, mean ± s.e.m. from 5 independent experiments) and protein (C). Total β-tubulin served as loading control. (D) Quantification of kinin B2 receptor by densitometry normalized to total β-tubulin. Values correspond to the mean ± s.e.m. from 3 independent experiments. (E) Retinas of rats that were intravitreously injected 4 h earlier with PBS, BK, Vi, or BK combined with Vi were analyzed for kinin B1 receptor protein. Total knee extract from rats with Freund's adjuvant-induced arthritis (AT, arthritic tissue) was used as a positive control for B1 receptor expression. NS, not significant.
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Figure 1: Vasoinhibins prevent BK-induced increase in BRB permeability similarly to a kinin B2 receptor antagonist. (A) Evaluation of the Evans blue dye content in retinas of rats intravitreously injected 4 h earlier with PBS (Ctl), BK (1 nM), BK combined with either vasoinhibins (Vi, 1 μM) or Hoe-140 (Hoe, 3 μM), Vi alone, or Hoe alone. Values are mean ± s.e.m. normalized to the control (n = 8–16 per group; *P < 0.05). (B–D) Retinas of rats that were intravitreously injected 4 h earlier with PBS, BK, Vi, or BK combined with Vi were analyzed for kinin B2 receptor mRNA (B, mean ± s.e.m. from 5 independent experiments) and protein (C). Total β-tubulin served as loading control. (D) Quantification of kinin B2 receptor by densitometry normalized to total β-tubulin. Values correspond to the mean ± s.e.m. from 3 independent experiments. (E) Retinas of rats that were intravitreously injected 4 h earlier with PBS, BK, Vi, or BK combined with Vi were analyzed for kinin B1 receptor protein. Total knee extract from rats with Freund's adjuvant-induced arthritis (AT, arthritic tissue) was used as a positive control for B1 receptor expression. NS, not significant.

Mentions: The intravitreal injection of BK induced a 1.8-fold increase in the basal transport through the BRB compared to the PBS-injected eyes (Ctl) (Figure 1A). When coinjected with BK, vasoinhibins prevented the BK-induced increase in BRB permeability in a manner similar to that of the competitive kinin B2 receptor antagonist Hoe-140 (Figure 1A). Alone, vasoinhibins or Hoe-140 did not modify the transport through the BRB. Signaling through the kinin B2 receptor has been shown to be primarily controlled by short-term mechanisms including both receptor desensitization (Mathis et al., 1996) and internalization (Munoz and Leeb-Lundberg, 1992; Munoz et al., 1993), but it can also be regulated by changes in expression levels of the receptor (Nostramo et al., 2013). Thus, retinas of animals that were intravitreally injected with BK, vasoinhibins, or both were analyzed for B2 receptor mRNA and protein levels by real-time PCR and Western-blot, respectively. The retinal levels of B2 receptor mRNA (Figure 1B) and protein (Figure 1C) were not different between PBS-, BK-, Vi-, and (BK + Vi)-injected eyes. Figure 1D shows quantification of B2 receptor after normalization for the amount of β-tubulin on the gel. Treatment with BK has been shown to induce the kinin B1 receptor (Phagoo et al., 1999). However, in addition to being absent in rat retina under physiological conditions, the B1 receptor is also absent after BK injection, combined or not with vasoinhibins (Figure 1E). Total knee extract from rats subjected to the adjuvant model of inflammatory arthritis for 21 days (Adan et al., 2013) was used as a positive control for B1 receptor expression. Notably, the band detected in arthritic tissue (AT) with the polyclonal anti-B1 receptor antibody migrates at an apparent molecular weight of 35 kDa, the molecular mass of the B1 receptor (see http://datasheets.scbt.com/sc-15048.pdf), thus validating the efficacy of the Western blot detection. These data indicate that vasoinhibins mitigate the BK-mediated increase in BRB permeability, that this results from kinin B2, but not B1, receptor activation, and that vasoinhibins do not change the amount of B2 receptor.


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 prevent BK-induced increase in BRB permeability similarly to a kinin B2 receptor antagonist. (A) Evaluation of the Evans blue dye content in retinas of rats intravitreously injected 4 h earlier with PBS (Ctl), BK (1 nM), BK combined with either vasoinhibins (Vi, 1 μM) or Hoe-140 (Hoe, 3 μM), Vi alone, or Hoe alone. Values are mean ± s.e.m. normalized to the control (n = 8–16 per group; *P < 0.05). (B–D) Retinas of rats that were intravitreously injected 4 h earlier with PBS, BK, Vi, or BK combined with Vi were analyzed for kinin B2 receptor mRNA (B, mean ± s.e.m. from 5 independent experiments) and protein (C). Total β-tubulin served as loading control. (D) Quantification of kinin B2 receptor by densitometry normalized to total β-tubulin. Values correspond to the mean ± s.e.m. from 3 independent experiments. (E) Retinas of rats that were intravitreously injected 4 h earlier with PBS, BK, Vi, or BK combined with Vi were analyzed for kinin B1 receptor protein. Total knee extract from rats with Freund's adjuvant-induced arthritis (AT, arthritic tissue) was used as a positive control for B1 receptor expression. NS, not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Vasoinhibins prevent BK-induced increase in BRB permeability similarly to a kinin B2 receptor antagonist. (A) Evaluation of the Evans blue dye content in retinas of rats intravitreously injected 4 h earlier with PBS (Ctl), BK (1 nM), BK combined with either vasoinhibins (Vi, 1 μM) or Hoe-140 (Hoe, 3 μM), Vi alone, or Hoe alone. Values are mean ± s.e.m. normalized to the control (n = 8–16 per group; *P < 0.05). (B–D) Retinas of rats that were intravitreously injected 4 h earlier with PBS, BK, Vi, or BK combined with Vi were analyzed for kinin B2 receptor mRNA (B, mean ± s.e.m. from 5 independent experiments) and protein (C). Total β-tubulin served as loading control. (D) Quantification of kinin B2 receptor by densitometry normalized to total β-tubulin. Values correspond to the mean ± s.e.m. from 3 independent experiments. (E) Retinas of rats that were intravitreously injected 4 h earlier with PBS, BK, Vi, or BK combined with Vi were analyzed for kinin B1 receptor protein. Total knee extract from rats with Freund's adjuvant-induced arthritis (AT, arthritic tissue) was used as a positive control for B1 receptor expression. NS, not significant.
Mentions: The intravitreal injection of BK induced a 1.8-fold increase in the basal transport through the BRB compared to the PBS-injected eyes (Ctl) (Figure 1A). When coinjected with BK, vasoinhibins prevented the BK-induced increase in BRB permeability in a manner similar to that of the competitive kinin B2 receptor antagonist Hoe-140 (Figure 1A). Alone, vasoinhibins or Hoe-140 did not modify the transport through the BRB. Signaling through the kinin B2 receptor has been shown to be primarily controlled by short-term mechanisms including both receptor desensitization (Mathis et al., 1996) and internalization (Munoz and Leeb-Lundberg, 1992; Munoz et al., 1993), but it can also be regulated by changes in expression levels of the receptor (Nostramo et al., 2013). Thus, retinas of animals that were intravitreally injected with BK, vasoinhibins, or both were analyzed for B2 receptor mRNA and protein levels by real-time PCR and Western-blot, respectively. The retinal levels of B2 receptor mRNA (Figure 1B) and protein (Figure 1C) were not different between PBS-, BK-, Vi-, and (BK + Vi)-injected eyes. Figure 1D shows quantification of B2 receptor after normalization for the amount of β-tubulin on the gel. Treatment with BK has been shown to induce the kinin B1 receptor (Phagoo et al., 1999). However, in addition to being absent in rat retina under physiological conditions, the B1 receptor is also absent after BK injection, combined or not with vasoinhibins (Figure 1E). Total knee extract from rats subjected to the adjuvant model of inflammatory arthritis for 21 days (Adan et al., 2013) was used as a positive control for B1 receptor expression. Notably, the band detected in arthritic tissue (AT) with the polyclonal anti-B1 receptor antibody migrates at an apparent molecular weight of 35 kDa, the molecular mass of the B1 receptor (see http://datasheets.scbt.com/sc-15048.pdf), thus validating the efficacy of the Western blot detection. These data indicate that vasoinhibins mitigate the BK-mediated increase in BRB permeability, that this results from kinin B2, but not B1, receptor activation, and that vasoinhibins do not change the amount of B2 receptor.

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