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TNF-α signals through PKCζ/NF-κB to alter the tight junction complex and increase retinal endothelial cell permeability.

Aveleira CA, Lin CM, Abcouwer SF, Ambrósio AF, Antonetti DA - Diabetes (2010)

Bottom Line: TNF-α decreased the protein and mRNA content of the tight junction proteins ZO-1 and claudin-5 and altered the cellular localization of these tight junction proteins.Preventing NF-κB activation with an inhibitor κB kinase (IKK) chemical inhibitor or adenoviral overexpression of inhibitor κB alpha (IκBα) reduced TNF-α-stimulated permeability.These results suggest that PKCζ may provide a specific therapeutic target for the prevention of vascular permeability in retinal diseases characterized by elevated TNF-α, including diabetic retinopathy.

View Article: PubMed Central - PubMed

Affiliation: Centre of Ophthalmology and Vision Sciences, Institute of Biomedical Research in Light and Image, Faculty of Medicine, University of Coimbra, Coimbra, Portugal. caveleira@ibili.uc.pt

ABSTRACT

Objective: Tumor necrosis factor-α (TNF-α) and interleukin-1 beta (IL-1β) are elevated in the vitreous of diabetic patients and in retinas of diabetic rats associated with increased retinal vascular permeability. However, the molecular mechanisms underlying retinal vascular permeability induced by these cytokines are poorly understood. In this study, the effects of IL-1β and TNF-α on retinal endothelial cell permeability were compared and the molecular mechanisms by which TNF-α increases cell permeability were elucidated.

Research design and methods: Cytokine-induced retinal vascular permeability was measured in bovine retinal endothelial cells (BRECs) and rat retinas. Western blotting, quantitative real-time PCR, and immunocytochemistry were performed to determine tight junction protein expression and localization.

Results: IL-1β and TNF-α increased BREC permeability, and TNF-α was more potent. TNF-α decreased the protein and mRNA content of the tight junction proteins ZO-1 and claudin-5 and altered the cellular localization of these tight junction proteins. Dexamethasone prevented TNF-α-induced cell permeability through glucocorticoid receptor transactivation and nuclear factor-kappaB (NF-κB) transrepression. Preventing NF-κB activation with an inhibitor κB kinase (IKK) chemical inhibitor or adenoviral overexpression of inhibitor κB alpha (IκBα) reduced TNF-α-stimulated permeability. Finally, inhibiting protein kinase C zeta (PKCζ) using both a peptide and a novel chemical inhibitor reduced NF-κB activation and completely prevented the alterations in the tight junction complex and cell permeability induced by TNF-α in cell culture and rat retinas.

Conclusions: These results suggest that PKCζ may provide a specific therapeutic target for the prevention of vascular permeability in retinal diseases characterized by elevated TNF-α, including diabetic retinopathy.

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Related in: MedlinePlus

PKCζI-1 prevents TNF-α–induced retinal vascular permeability in vivo. Animals' eyes were injected with PBS with 0.1% BSA, TNF-α (10 ng); PKCζI-1 (280 ng); or with both PKCζI-1 and TNF-α. A: Evans blue leakage was evaluated 24 h after intravitreous injections. The results represent the mean ± SEM (n = 7–8 animals per group) and are expressed relative to control (Ctrl; PBS-injected eyes). **P < 0.01, significantly different from control; ##P < 0.01, significantly different from TNF-α, as determined by ANOVA followed by Bonferroni post hoc test. B: PKCζI-1 prevents the alterations in tight junction proteins induced by TNF-α in vivo. Whole retinas were immunolabeled for ZO-1 and occludin 4 h after injection. Images were obtained on a Leica TCS SP2 AOBS confocal microscope and are presented as a maximum projection. Arrows indicate loss and/or discontinuous cell border staining. Scale bar, 25 μm. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 8: PKCζI-1 prevents TNF-α–induced retinal vascular permeability in vivo. Animals' eyes were injected with PBS with 0.1% BSA, TNF-α (10 ng); PKCζI-1 (280 ng); or with both PKCζI-1 and TNF-α. A: Evans blue leakage was evaluated 24 h after intravitreous injections. The results represent the mean ± SEM (n = 7–8 animals per group) and are expressed relative to control (Ctrl; PBS-injected eyes). **P < 0.01, significantly different from control; ##P < 0.01, significantly different from TNF-α, as determined by ANOVA followed by Bonferroni post hoc test. B: PKCζI-1 prevents the alterations in tight junction proteins induced by TNF-α in vivo. Whole retinas were immunolabeled for ZO-1 and occludin 4 h after injection. Images were obtained on a Leica TCS SP2 AOBS confocal microscope and are presented as a maximum projection. Arrows indicate loss and/or discontinuous cell border staining. Scale bar, 25 μm. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: The effect of PKCζI-1 on TNF-α–induced BRB permeability was evaluated in vivo by the Evans blue assay. The injection of TNF-α in the vitreous induced the accumulation of Evans blue (20.30 ± 2.46 μl plasma/g/h) as compared with PBS-injected animals (10.25 ± 2.48 μl plasma/g/h; Fig. 8A). PKCζI-1 alone had no effect on the accumulation of Evans blue but completely prevented TNF-α–induced accumulation of the dye. To determine the effect of TNF-α injection and PKCζ inhibition on retinal vascular tight junction organization, retina whole flat mounts were immunolabeled for ZO-1 and occludin proteins after injection of the cytokine or cytokine with PKCζI-1. In PBS-injected rat eyes, ZO-1 and occludin immunoreactivities were intense and localized at the junctions of the cell membranes of endothelial cells in retinal vessels. In TNF-α-injected eyes, changes in ZO-1 immunostaining were particularly evident, which became markedly reduced and intermittently absent from the cell borders. These alterations were prevented by cotreatment with PKCζI-1 (Fig. 8B). Consistent with the results obtained with BRECs in culture, the occludin content was not reduced with TNF-α but displayed regions with disorganized cell-border labeling that were reversed by PKCζI-1 treatment.


TNF-α signals through PKCζ/NF-κB to alter the tight junction complex and increase retinal endothelial cell permeability.

Aveleira CA, Lin CM, Abcouwer SF, Ambrósio AF, Antonetti DA - Diabetes (2010)

PKCζI-1 prevents TNF-α–induced retinal vascular permeability in vivo. Animals' eyes were injected with PBS with 0.1% BSA, TNF-α (10 ng); PKCζI-1 (280 ng); or with both PKCζI-1 and TNF-α. A: Evans blue leakage was evaluated 24 h after intravitreous injections. The results represent the mean ± SEM (n = 7–8 animals per group) and are expressed relative to control (Ctrl; PBS-injected eyes). **P < 0.01, significantly different from control; ##P < 0.01, significantly different from TNF-α, as determined by ANOVA followed by Bonferroni post hoc test. B: PKCζI-1 prevents the alterations in tight junction proteins induced by TNF-α in vivo. Whole retinas were immunolabeled for ZO-1 and occludin 4 h after injection. Images were obtained on a Leica TCS SP2 AOBS confocal microscope and are presented as a maximum projection. Arrows indicate loss and/or discontinuous cell border staining. Scale bar, 25 μm. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 8: PKCζI-1 prevents TNF-α–induced retinal vascular permeability in vivo. Animals' eyes were injected with PBS with 0.1% BSA, TNF-α (10 ng); PKCζI-1 (280 ng); or with both PKCζI-1 and TNF-α. A: Evans blue leakage was evaluated 24 h after intravitreous injections. The results represent the mean ± SEM (n = 7–8 animals per group) and are expressed relative to control (Ctrl; PBS-injected eyes). **P < 0.01, significantly different from control; ##P < 0.01, significantly different from TNF-α, as determined by ANOVA followed by Bonferroni post hoc test. B: PKCζI-1 prevents the alterations in tight junction proteins induced by TNF-α in vivo. Whole retinas were immunolabeled for ZO-1 and occludin 4 h after injection. Images were obtained on a Leica TCS SP2 AOBS confocal microscope and are presented as a maximum projection. Arrows indicate loss and/or discontinuous cell border staining. Scale bar, 25 μm. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: The effect of PKCζI-1 on TNF-α–induced BRB permeability was evaluated in vivo by the Evans blue assay. The injection of TNF-α in the vitreous induced the accumulation of Evans blue (20.30 ± 2.46 μl plasma/g/h) as compared with PBS-injected animals (10.25 ± 2.48 μl plasma/g/h; Fig. 8A). PKCζI-1 alone had no effect on the accumulation of Evans blue but completely prevented TNF-α–induced accumulation of the dye. To determine the effect of TNF-α injection and PKCζ inhibition on retinal vascular tight junction organization, retina whole flat mounts were immunolabeled for ZO-1 and occludin proteins after injection of the cytokine or cytokine with PKCζI-1. In PBS-injected rat eyes, ZO-1 and occludin immunoreactivities were intense and localized at the junctions of the cell membranes of endothelial cells in retinal vessels. In TNF-α-injected eyes, changes in ZO-1 immunostaining were particularly evident, which became markedly reduced and intermittently absent from the cell borders. These alterations were prevented by cotreatment with PKCζI-1 (Fig. 8B). Consistent with the results obtained with BRECs in culture, the occludin content was not reduced with TNF-α but displayed regions with disorganized cell-border labeling that were reversed by PKCζI-1 treatment.

Bottom Line: TNF-α decreased the protein and mRNA content of the tight junction proteins ZO-1 and claudin-5 and altered the cellular localization of these tight junction proteins.Preventing NF-κB activation with an inhibitor κB kinase (IKK) chemical inhibitor or adenoviral overexpression of inhibitor κB alpha (IκBα) reduced TNF-α-stimulated permeability.These results suggest that PKCζ may provide a specific therapeutic target for the prevention of vascular permeability in retinal diseases characterized by elevated TNF-α, including diabetic retinopathy.

View Article: PubMed Central - PubMed

Affiliation: Centre of Ophthalmology and Vision Sciences, Institute of Biomedical Research in Light and Image, Faculty of Medicine, University of Coimbra, Coimbra, Portugal. caveleira@ibili.uc.pt

ABSTRACT

Objective: Tumor necrosis factor-α (TNF-α) and interleukin-1 beta (IL-1β) are elevated in the vitreous of diabetic patients and in retinas of diabetic rats associated with increased retinal vascular permeability. However, the molecular mechanisms underlying retinal vascular permeability induced by these cytokines are poorly understood. In this study, the effects of IL-1β and TNF-α on retinal endothelial cell permeability were compared and the molecular mechanisms by which TNF-α increases cell permeability were elucidated.

Research design and methods: Cytokine-induced retinal vascular permeability was measured in bovine retinal endothelial cells (BRECs) and rat retinas. Western blotting, quantitative real-time PCR, and immunocytochemistry were performed to determine tight junction protein expression and localization.

Results: IL-1β and TNF-α increased BREC permeability, and TNF-α was more potent. TNF-α decreased the protein and mRNA content of the tight junction proteins ZO-1 and claudin-5 and altered the cellular localization of these tight junction proteins. Dexamethasone prevented TNF-α-induced cell permeability through glucocorticoid receptor transactivation and nuclear factor-kappaB (NF-κB) transrepression. Preventing NF-κB activation with an inhibitor κB kinase (IKK) chemical inhibitor or adenoviral overexpression of inhibitor κB alpha (IκBα) reduced TNF-α-stimulated permeability. Finally, inhibiting protein kinase C zeta (PKCζ) using both a peptide and a novel chemical inhibitor reduced NF-κB activation and completely prevented the alterations in the tight junction complex and cell permeability induced by TNF-α in cell culture and rat retinas.

Conclusions: These results suggest that PKCζ may provide a specific therapeutic target for the prevention of vascular permeability in retinal diseases characterized by elevated TNF-α, including diabetic retinopathy.

Show MeSH
Related in: MedlinePlus