Limits...
PEDF regulates vascular permeability by a γ-secretase-mediated pathway.

Cai J, Wu L, Qi X, Li Calzi S, Caballero S, Shaw L, Ruan Q, Grant MB, Boulton ME - PLoS ONE (2011)

Bottom Line: In selected experiments the effect of hypoxia on junctional integrity was also assessed.Although hypoxia increased VEGFR expression there was a significant dissociation of VEGFR from AJ proteins.In conclusion, PEDF regulates VEGF-induced vascular permeability via a novel γ-secretase dependent pathway and targeting downstream effectors of PEDF action may represent a promising therapeutic strategy for preventing or ameliorating increased vascular permeability.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
Increased vascular permeability is an inciting event in many vascular complications including diabetic retinopathy. We have previously reported that pigment epithelium-derived factor (PEDF) is able to inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis through a novel γ-secretase-dependent pathway. In this study, we asked whether inhibition of VEGF-induced permeability by PEDF is also γ-secretase-mediated and to dissect the potential mechanisms involved. Vascular permeability was assessed in vitro by measuring transendothelial resistance and paracellular permeability to dextran and in vivo by following leakage of intravenous FITC-labelled albumin into the retina in the presence or absence of VEGF and PEDF in varying combinations. Experiments were undertaken in the presence or absence of a γ-secretase inhibitor. To assess junctional integrity immunohistochemistry for the adherens junction (AJ) proteins, VE-cadherin and β-catenin, and the tight junction (TJ) protein, claudin-5 was undertaken using cultured cells and flat mount retinas. Protein expression and the association between AJ proteins, VEGF receptors (VEGFRs) and γ-secretase constituents were determined by immunoprecipitation and Western Blot analysis. In selected experiments the effect of hypoxia on junctional integrity was also assessed. PEDF inhibition of VEGF-induced permeability, both in cultured microvascular endothelial cell monolayers and in vivo in the mouse retinal vasculature, is mediated by γ-secretase. PEDF acted by a) preventing dissociation of AJ and TJ proteins and b) regulating both the association of VEGF receptors with AJ proteins and the subsequent phosphorylation of the AJ proteins, VE-cadherin and β-catenin. Association of γ-secretase with AJ proteins appears to be critical in the regulation of vascular permeability. Although hypoxia increased VEGFR expression there was a significant dissociation of VEGFR from AJ proteins. In conclusion, PEDF regulates VEGF-induced vascular permeability via a novel γ-secretase dependent pathway and targeting downstream effectors of PEDF action may represent a promising therapeutic strategy for preventing or ameliorating increased vascular permeability.

Show MeSH

Related in: MedlinePlus

PEDF prevents VEGF-induced dissociation of endothelial AJs and TJs.Representative immunofluorescent images of confluent cultures of microvascular endothelial cells treated with vehicle (unstimulated), VEGFA alone, PEDF alone, simultaneous VEGFA+PEDF, PEDF 6 hours post VEGF and PEDF 6 hours post VEGF+γ-secretase inhibitor (γ-SI) (n = 4 independent experiments). VEGFA and PEDF were used at 100 ng/ml and γ-secretase inhibitor at 1 nM. (a) Cultures were triple stained for VE-cadherin (red), claudin-5 (green) and nuclei (DAPI, blue) and assessed at different times over 24 hours using confocal microscopy. Merged images are shown with colocalization of VE-cadherin and claudin-5 appearing as yellow. (b) The effect of PEDF on β-catenin using the conditions described in (A) (n = 4). Scale bar = 100 µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3117873&req=5

pone-0021164-g003: PEDF prevents VEGF-induced dissociation of endothelial AJs and TJs.Representative immunofluorescent images of confluent cultures of microvascular endothelial cells treated with vehicle (unstimulated), VEGFA alone, PEDF alone, simultaneous VEGFA+PEDF, PEDF 6 hours post VEGF and PEDF 6 hours post VEGF+γ-secretase inhibitor (γ-SI) (n = 4 independent experiments). VEGFA and PEDF were used at 100 ng/ml and γ-secretase inhibitor at 1 nM. (a) Cultures were triple stained for VE-cadherin (red), claudin-5 (green) and nuclei (DAPI, blue) and assessed at different times over 24 hours using confocal microscopy. Merged images are shown with colocalization of VE-cadherin and claudin-5 appearing as yellow. (b) The effect of PEDF on β-catenin using the conditions described in (A) (n = 4). Scale bar = 100 µm.

Mentions: Immunohistochemistry demonstrated lateral membrane localization of VE-cadherin and claudin-5 in endothelial cell monolayers and this was unaffected by addition of PEDF alone (Fig. 2) or γ-secretase inhibitor alone. The vehicle control remained identical to VE-cadherin and Claudin-5 staining at time 0 throughout the time course of the experiment. However, VEGF alone led to a rapid disassociation of the cell-cell contacts and a major loss of staining for VE-cadherin at the junctional complexes within 15 minutes and reduced staining for claudin 5 observed at 1 and 12 hours post treatment (Fig. 2). Addition of PEDF in combination with VEGF (data not shown), or PEDF 6 hours post VEGF treatment, prevented the VEGF-induced dissociation of lateral junctional complexes (Fig. 3). However, γ-secretase inhibition, blocked the inhibitory effect of PEDF on VEGF-induced junctional changes. β-catenin, which is associated with the integrity of VE-cadherin in adherens junction [8], was dissociated by VEGF and translocated to both the cytosolic and nuclear compartments and this VEGF-induced dissociation was prevented by PEDF (Fig. 3). Inhibition of γ-secretase prevented the PEDF-induced effect on VEGF dissociation of β-catenin.


PEDF regulates vascular permeability by a γ-secretase-mediated pathway.

Cai J, Wu L, Qi X, Li Calzi S, Caballero S, Shaw L, Ruan Q, Grant MB, Boulton ME - PLoS ONE (2011)

PEDF prevents VEGF-induced dissociation of endothelial AJs and TJs.Representative immunofluorescent images of confluent cultures of microvascular endothelial cells treated with vehicle (unstimulated), VEGFA alone, PEDF alone, simultaneous VEGFA+PEDF, PEDF 6 hours post VEGF and PEDF 6 hours post VEGF+γ-secretase inhibitor (γ-SI) (n = 4 independent experiments). VEGFA and PEDF were used at 100 ng/ml and γ-secretase inhibitor at 1 nM. (a) Cultures were triple stained for VE-cadherin (red), claudin-5 (green) and nuclei (DAPI, blue) and assessed at different times over 24 hours using confocal microscopy. Merged images are shown with colocalization of VE-cadherin and claudin-5 appearing as yellow. (b) The effect of PEDF on β-catenin using the conditions described in (A) (n = 4). Scale bar = 100 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3117873&req=5

pone-0021164-g003: PEDF prevents VEGF-induced dissociation of endothelial AJs and TJs.Representative immunofluorescent images of confluent cultures of microvascular endothelial cells treated with vehicle (unstimulated), VEGFA alone, PEDF alone, simultaneous VEGFA+PEDF, PEDF 6 hours post VEGF and PEDF 6 hours post VEGF+γ-secretase inhibitor (γ-SI) (n = 4 independent experiments). VEGFA and PEDF were used at 100 ng/ml and γ-secretase inhibitor at 1 nM. (a) Cultures were triple stained for VE-cadherin (red), claudin-5 (green) and nuclei (DAPI, blue) and assessed at different times over 24 hours using confocal microscopy. Merged images are shown with colocalization of VE-cadherin and claudin-5 appearing as yellow. (b) The effect of PEDF on β-catenin using the conditions described in (A) (n = 4). Scale bar = 100 µm.
Mentions: Immunohistochemistry demonstrated lateral membrane localization of VE-cadherin and claudin-5 in endothelial cell monolayers and this was unaffected by addition of PEDF alone (Fig. 2) or γ-secretase inhibitor alone. The vehicle control remained identical to VE-cadherin and Claudin-5 staining at time 0 throughout the time course of the experiment. However, VEGF alone led to a rapid disassociation of the cell-cell contacts and a major loss of staining for VE-cadherin at the junctional complexes within 15 minutes and reduced staining for claudin 5 observed at 1 and 12 hours post treatment (Fig. 2). Addition of PEDF in combination with VEGF (data not shown), or PEDF 6 hours post VEGF treatment, prevented the VEGF-induced dissociation of lateral junctional complexes (Fig. 3). However, γ-secretase inhibition, blocked the inhibitory effect of PEDF on VEGF-induced junctional changes. β-catenin, which is associated with the integrity of VE-cadherin in adherens junction [8], was dissociated by VEGF and translocated to both the cytosolic and nuclear compartments and this VEGF-induced dissociation was prevented by PEDF (Fig. 3). Inhibition of γ-secretase prevented the PEDF-induced effect on VEGF dissociation of β-catenin.

Bottom Line: In selected experiments the effect of hypoxia on junctional integrity was also assessed.Although hypoxia increased VEGFR expression there was a significant dissociation of VEGFR from AJ proteins.In conclusion, PEDF regulates VEGF-induced vascular permeability via a novel γ-secretase dependent pathway and targeting downstream effectors of PEDF action may represent a promising therapeutic strategy for preventing or ameliorating increased vascular permeability.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
Increased vascular permeability is an inciting event in many vascular complications including diabetic retinopathy. We have previously reported that pigment epithelium-derived factor (PEDF) is able to inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis through a novel γ-secretase-dependent pathway. In this study, we asked whether inhibition of VEGF-induced permeability by PEDF is also γ-secretase-mediated and to dissect the potential mechanisms involved. Vascular permeability was assessed in vitro by measuring transendothelial resistance and paracellular permeability to dextran and in vivo by following leakage of intravenous FITC-labelled albumin into the retina in the presence or absence of VEGF and PEDF in varying combinations. Experiments were undertaken in the presence or absence of a γ-secretase inhibitor. To assess junctional integrity immunohistochemistry for the adherens junction (AJ) proteins, VE-cadherin and β-catenin, and the tight junction (TJ) protein, claudin-5 was undertaken using cultured cells and flat mount retinas. Protein expression and the association between AJ proteins, VEGF receptors (VEGFRs) and γ-secretase constituents were determined by immunoprecipitation and Western Blot analysis. In selected experiments the effect of hypoxia on junctional integrity was also assessed. PEDF inhibition of VEGF-induced permeability, both in cultured microvascular endothelial cell monolayers and in vivo in the mouse retinal vasculature, is mediated by γ-secretase. PEDF acted by a) preventing dissociation of AJ and TJ proteins and b) regulating both the association of VEGF receptors with AJ proteins and the subsequent phosphorylation of the AJ proteins, VE-cadherin and β-catenin. Association of γ-secretase with AJ proteins appears to be critical in the regulation of vascular permeability. Although hypoxia increased VEGFR expression there was a significant dissociation of VEGFR from AJ proteins. In conclusion, PEDF regulates VEGF-induced vascular permeability via a novel γ-secretase dependent pathway and targeting downstream effectors of PEDF action may represent a promising therapeutic strategy for preventing or ameliorating increased vascular permeability.

Show MeSH
Related in: MedlinePlus