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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.

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PEDF maintains the association between AJ proteins and VEGF receptors in VEGF treated cells.Confluent retinal endothelial cells were either unstimulated (1) treated with VEGF alone (2), PEDF alone (3), VEGF followed by PEDF 6 hours later without (4) and with (5) γ-secretase inhibitor for 24 hours. VEGFA and PEDF were used at 100 ng/ml and γ-secretase inhibitor at 1 nM. Total cell lysates were split into four equal portions and immunoprecipitated with anti-VE-cadherin, anti-β-catenin, anti-VEGFR1-C-terminus and anti-VEGFR2-C-terminus, and then subsequent Western blot analyzed using these four antibodies, respectively. (a) A panel of representative Western blots. (b) Band intensities of replicate experiments (n = 4 independent experiments) were quantified as described in the Methods and regression analysis undertaken to assess the association of these proteins.
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pone-0021164-g006: PEDF maintains the association between AJ proteins and VEGF receptors in VEGF treated cells.Confluent retinal endothelial cells were either unstimulated (1) treated with VEGF alone (2), PEDF alone (3), VEGF followed by PEDF 6 hours later without (4) and with (5) γ-secretase inhibitor for 24 hours. VEGFA and PEDF were used at 100 ng/ml and γ-secretase inhibitor at 1 nM. Total cell lysates were split into four equal portions and immunoprecipitated with anti-VE-cadherin, anti-β-catenin, anti-VEGFR1-C-terminus and anti-VEGFR2-C-terminus, and then subsequent Western blot analyzed using these four antibodies, respectively. (a) A panel of representative Western blots. (b) Band intensities of replicate experiments (n = 4 independent experiments) were quantified as described in the Methods and regression analysis undertaken to assess the association of these proteins.

Mentions: Immunoprecipitation and Western blot demonstrated a strong, but dynamic, association between VE cadherin, β-catenin and full length VEGFRs 1 and 2 (Fig. 6). In the untreated cells, VEGFR1 demonstrated stronger association with VE-cadherin and β-catenin than VEGFR2. PEDF alone did not affect this association. However, a decreased VEGFR-1 association with VE-cadherin and β-catenin was observed after one-hour VEGF treatment while the association of VEGFR-2 with VE-cadherin/β-catenin remained unchanged, thus reducing the VEGFR-1∶VEGFR-2 ratio. By 6 hr, sustained presence of VEGF further reduced VEGFR-1 association with VE-cadherin/β-catenin but increased VEGFR-2 which led to a further reduction in the VEGFR-1∶VEGFR-2 ratio. By 24r, VEGF treatment not only caused a dissociation of the VE-cadherin/β-catenin complex, but also an almost complete loss of both VEGFR-1 and VEGFR-2 association with VE-cadherin or β-catenin. Taken together, these data suggest that the ratio of VEGFR1∶VEGFR2 may be important in the integrity of AJs. VEGF-induced VEGFR/VE-cadherin/β-catenin dissociation could be blocked by adding PEDF and this could be reversed by γ-secretase inhibition (Fig. 6).


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 maintains the association between AJ proteins and VEGF receptors in VEGF treated cells.Confluent retinal endothelial cells were either unstimulated (1) treated with VEGF alone (2), PEDF alone (3), VEGF followed by PEDF 6 hours later without (4) and with (5) γ-secretase inhibitor for 24 hours. VEGFA and PEDF were used at 100 ng/ml and γ-secretase inhibitor at 1 nM. Total cell lysates were split into four equal portions and immunoprecipitated with anti-VE-cadherin, anti-β-catenin, anti-VEGFR1-C-terminus and anti-VEGFR2-C-terminus, and then subsequent Western blot analyzed using these four antibodies, respectively. (a) A panel of representative Western blots. (b) Band intensities of replicate experiments (n = 4 independent experiments) were quantified as described in the Methods and regression analysis undertaken to assess the association of these proteins.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3117873&req=5

pone-0021164-g006: PEDF maintains the association between AJ proteins and VEGF receptors in VEGF treated cells.Confluent retinal endothelial cells were either unstimulated (1) treated with VEGF alone (2), PEDF alone (3), VEGF followed by PEDF 6 hours later without (4) and with (5) γ-secretase inhibitor for 24 hours. VEGFA and PEDF were used at 100 ng/ml and γ-secretase inhibitor at 1 nM. Total cell lysates were split into four equal portions and immunoprecipitated with anti-VE-cadherin, anti-β-catenin, anti-VEGFR1-C-terminus and anti-VEGFR2-C-terminus, and then subsequent Western blot analyzed using these four antibodies, respectively. (a) A panel of representative Western blots. (b) Band intensities of replicate experiments (n = 4 independent experiments) were quantified as described in the Methods and regression analysis undertaken to assess the association of these proteins.
Mentions: Immunoprecipitation and Western blot demonstrated a strong, but dynamic, association between VE cadherin, β-catenin and full length VEGFRs 1 and 2 (Fig. 6). In the untreated cells, VEGFR1 demonstrated stronger association with VE-cadherin and β-catenin than VEGFR2. PEDF alone did not affect this association. However, a decreased VEGFR-1 association with VE-cadherin and β-catenin was observed after one-hour VEGF treatment while the association of VEGFR-2 with VE-cadherin/β-catenin remained unchanged, thus reducing the VEGFR-1∶VEGFR-2 ratio. By 6 hr, sustained presence of VEGF further reduced VEGFR-1 association with VE-cadherin/β-catenin but increased VEGFR-2 which led to a further reduction in the VEGFR-1∶VEGFR-2 ratio. By 24r, VEGF treatment not only caused a dissociation of the VE-cadherin/β-catenin complex, but also an almost complete loss of both VEGFR-1 and VEGFR-2 association with VE-cadherin or β-catenin. Taken together, these data suggest that the ratio of VEGFR1∶VEGFR2 may be important in the integrity of AJs. VEGF-induced VEGFR/VE-cadherin/β-catenin dissociation could be blocked by adding PEDF and this could be reversed by γ-secretase inhibition (Fig. 6).

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