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The integrin antagonist cilengitide activates alphaVbeta3, disrupts VE-cadherin localization at cell junctions and enhances permeability in endothelial cells.

Alghisi GC, Ponsonnet L, Rüegg C - PLoS ONE (2009)

Bottom Line: Angiogenic endothelial cells express multiple integrins, in particular those of the beta1 family, and little is known on the effect of cilengitide on endothelial cells expressing alphaVbeta3 but adhering through beta1 integrins.Through morphological, biochemical, pharmacological and functional approaches we investigated the effect of cilengitide on alphaVbeta3-expressing human umbilical vein endothelial cells (HUVEC) cultured on the beta1 ligands fibronectin and collagen I.These effects are potentially relevant to the clinical use of cilengitide as anticancer agent.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Oncology, Centre Pluridisciplinaire d'Oncologie (CePO), Faculty of Biology and Medicine, University of Lausanne, and NCCR Molecular Oncology, ISREC, Epalinges, Switzerland.

ABSTRACT
Cilengitide is a high-affinity cyclic pentapeptdic alphaV integrin antagonist previously reported to suppress angiogenesis by inducing anoikis of endothelial cells adhering through alphaVbeta3/alphaVbeta5 integrins. Angiogenic endothelial cells express multiple integrins, in particular those of the beta1 family, and little is known on the effect of cilengitide on endothelial cells expressing alphaVbeta3 but adhering through beta1 integrins. Through morphological, biochemical, pharmacological and functional approaches we investigated the effect of cilengitide on alphaVbeta3-expressing human umbilical vein endothelial cells (HUVEC) cultured on the beta1 ligands fibronectin and collagen I. We show that cilengitide activated cell surface alphaVbeta3, stimulated phosphorylation of FAK (Y(397) and Y(576/577)), Src (S(418)) and VE-cadherin (Y(658) and Y(731)), redistributed alphaVbeta3 at the cell periphery, caused disappearance of VE-cadherin from cellular junctions, increased the permeability of HUVEC monolayers and detached HUVEC adhering on low-density beta1 integrin ligands. Pharmacological inhibition of Src kinase activity fully prevented cilengitide-induced phosphorylation of Src, FAK and VE-cadherin, and redistribution of alphaVbeta3 and VE-cadherin and partially prevented increased permeability, but did not prevent HUVEC detachment from low-density matrices. Taken together, these observations reveal a previously unreported effect of cilengitide on endothelial cells namely its ability to elicit signaling events disrupting VE-cadherin localization at cellular contacts and to increase endothelial monolayer permeability. These effects are potentially relevant to the clinical use of cilengitide as anticancer agent.

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Cilengitide induces VE-cadherin loss form cellular junctions.(a) Confluent HUVEC plated on fibronectin, were exposed to cilengitide or EMD135981 (10 µM each) or VEGF (100 ng/ml) for 20 minutes and stained for VE-cadherin. Cilengitide and VEGF treatments disrupted VE-cadherin localization at cellular junctions, while EMD135981 showed no effect (n = 3). Optical magnification: 400×; Bar: 10 µm. (b) Confluent HUVEC plated on fibronectin or collagen I were exposed to cilengitide (10 µM each) for the indicated time and double stained for VE-cadherin and β3 integrin. Cilengitide disrupted VE-cadherin staining and promoted appearance of β3 at VE-cadherin-depleted cell-cell borders (arrows). Paralleling loss of VE-cadherin from cell-cell junctions, ‘gaps’ appeared in the monolayer (asterisks). (n = 4). Optical magnification: 400×; Bar: 10 µm. (c) Higher magnification (2× zooming in) of HUVEC cultures of the experiment shown in panel b to demonstrate rare co-localization of VE-cadherin and β3 integrin at cellular junctions upon cilengitide stimulation (arrowheads). (n = 4). Bars: 10 µm.
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pone-0004449-g002: Cilengitide induces VE-cadherin loss form cellular junctions.(a) Confluent HUVEC plated on fibronectin, were exposed to cilengitide or EMD135981 (10 µM each) or VEGF (100 ng/ml) for 20 minutes and stained for VE-cadherin. Cilengitide and VEGF treatments disrupted VE-cadherin localization at cellular junctions, while EMD135981 showed no effect (n = 3). Optical magnification: 400×; Bar: 10 µm. (b) Confluent HUVEC plated on fibronectin or collagen I were exposed to cilengitide (10 µM each) for the indicated time and double stained for VE-cadherin and β3 integrin. Cilengitide disrupted VE-cadherin staining and promoted appearance of β3 at VE-cadherin-depleted cell-cell borders (arrows). Paralleling loss of VE-cadherin from cell-cell junctions, ‘gaps’ appeared in the monolayer (asterisks). (n = 4). Optical magnification: 400×; Bar: 10 µm. (c) Higher magnification (2× zooming in) of HUVEC cultures of the experiment shown in panel b to demonstrate rare co-localization of VE-cadherin and β3 integrin at cellular junctions upon cilengitide stimulation (arrowheads). (n = 4). Bars: 10 µm.

Mentions: VE-cadherin is a major endothelial cell junctional molecule mediating cell–cell adhesion [25]. It has been previously reported that integrin ligation through multivalent fibronectin-coated beads disrupted VE-cadherin-containing adherens junctions in bovine aortic endothelial cells [26]. We therefore tested, whether αVβ3 ligation by monovalent cilengitide affected VE-cadherin localization. In confluent monolayers of HUVEC cultured for 18 hours on fibronectin or collagen I, VE-cadherin was localized at cell-cell contacts (Figure 2a and data not shown). Addition of cilengitide markedly disrupted VE-cadherin localization at cellular junctions, while the EMD135981 peptide was ineffective. Stimulation with VEGF also caused VE-cadherin disappearance from cellular junctions (Figure 2a), consistent with previous reports [27].


The integrin antagonist cilengitide activates alphaVbeta3, disrupts VE-cadherin localization at cell junctions and enhances permeability in endothelial cells.

Alghisi GC, Ponsonnet L, Rüegg C - PLoS ONE (2009)

Cilengitide induces VE-cadherin loss form cellular junctions.(a) Confluent HUVEC plated on fibronectin, were exposed to cilengitide or EMD135981 (10 µM each) or VEGF (100 ng/ml) for 20 minutes and stained for VE-cadherin. Cilengitide and VEGF treatments disrupted VE-cadherin localization at cellular junctions, while EMD135981 showed no effect (n = 3). Optical magnification: 400×; Bar: 10 µm. (b) Confluent HUVEC plated on fibronectin or collagen I were exposed to cilengitide (10 µM each) for the indicated time and double stained for VE-cadherin and β3 integrin. Cilengitide disrupted VE-cadherin staining and promoted appearance of β3 at VE-cadherin-depleted cell-cell borders (arrows). Paralleling loss of VE-cadherin from cell-cell junctions, ‘gaps’ appeared in the monolayer (asterisks). (n = 4). Optical magnification: 400×; Bar: 10 µm. (c) Higher magnification (2× zooming in) of HUVEC cultures of the experiment shown in panel b to demonstrate rare co-localization of VE-cadherin and β3 integrin at cellular junctions upon cilengitide stimulation (arrowheads). (n = 4). Bars: 10 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2636874&req=5

pone-0004449-g002: Cilengitide induces VE-cadherin loss form cellular junctions.(a) Confluent HUVEC plated on fibronectin, were exposed to cilengitide or EMD135981 (10 µM each) or VEGF (100 ng/ml) for 20 minutes and stained for VE-cadherin. Cilengitide and VEGF treatments disrupted VE-cadherin localization at cellular junctions, while EMD135981 showed no effect (n = 3). Optical magnification: 400×; Bar: 10 µm. (b) Confluent HUVEC plated on fibronectin or collagen I were exposed to cilengitide (10 µM each) for the indicated time and double stained for VE-cadherin and β3 integrin. Cilengitide disrupted VE-cadherin staining and promoted appearance of β3 at VE-cadherin-depleted cell-cell borders (arrows). Paralleling loss of VE-cadherin from cell-cell junctions, ‘gaps’ appeared in the monolayer (asterisks). (n = 4). Optical magnification: 400×; Bar: 10 µm. (c) Higher magnification (2× zooming in) of HUVEC cultures of the experiment shown in panel b to demonstrate rare co-localization of VE-cadherin and β3 integrin at cellular junctions upon cilengitide stimulation (arrowheads). (n = 4). Bars: 10 µm.
Mentions: VE-cadherin is a major endothelial cell junctional molecule mediating cell–cell adhesion [25]. It has been previously reported that integrin ligation through multivalent fibronectin-coated beads disrupted VE-cadherin-containing adherens junctions in bovine aortic endothelial cells [26]. We therefore tested, whether αVβ3 ligation by monovalent cilengitide affected VE-cadherin localization. In confluent monolayers of HUVEC cultured for 18 hours on fibronectin or collagen I, VE-cadherin was localized at cell-cell contacts (Figure 2a and data not shown). Addition of cilengitide markedly disrupted VE-cadherin localization at cellular junctions, while the EMD135981 peptide was ineffective. Stimulation with VEGF also caused VE-cadherin disappearance from cellular junctions (Figure 2a), consistent with previous reports [27].

Bottom Line: Angiogenic endothelial cells express multiple integrins, in particular those of the beta1 family, and little is known on the effect of cilengitide on endothelial cells expressing alphaVbeta3 but adhering through beta1 integrins.Through morphological, biochemical, pharmacological and functional approaches we investigated the effect of cilengitide on alphaVbeta3-expressing human umbilical vein endothelial cells (HUVEC) cultured on the beta1 ligands fibronectin and collagen I.These effects are potentially relevant to the clinical use of cilengitide as anticancer agent.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Oncology, Centre Pluridisciplinaire d'Oncologie (CePO), Faculty of Biology and Medicine, University of Lausanne, and NCCR Molecular Oncology, ISREC, Epalinges, Switzerland.

ABSTRACT
Cilengitide is a high-affinity cyclic pentapeptdic alphaV integrin antagonist previously reported to suppress angiogenesis by inducing anoikis of endothelial cells adhering through alphaVbeta3/alphaVbeta5 integrins. Angiogenic endothelial cells express multiple integrins, in particular those of the beta1 family, and little is known on the effect of cilengitide on endothelial cells expressing alphaVbeta3 but adhering through beta1 integrins. Through morphological, biochemical, pharmacological and functional approaches we investigated the effect of cilengitide on alphaVbeta3-expressing human umbilical vein endothelial cells (HUVEC) cultured on the beta1 ligands fibronectin and collagen I. We show that cilengitide activated cell surface alphaVbeta3, stimulated phosphorylation of FAK (Y(397) and Y(576/577)), Src (S(418)) and VE-cadherin (Y(658) and Y(731)), redistributed alphaVbeta3 at the cell periphery, caused disappearance of VE-cadherin from cellular junctions, increased the permeability of HUVEC monolayers and detached HUVEC adhering on low-density beta1 integrin ligands. Pharmacological inhibition of Src kinase activity fully prevented cilengitide-induced phosphorylation of Src, FAK and VE-cadherin, and redistribution of alphaVbeta3 and VE-cadherin and partially prevented increased permeability, but did not prevent HUVEC detachment from low-density matrices. Taken together, these observations reveal a previously unreported effect of cilengitide on endothelial cells namely its ability to elicit signaling events disrupting VE-cadherin localization at cellular contacts and to increase endothelial monolayer permeability. These effects are potentially relevant to the clinical use of cilengitide as anticancer agent.

Show MeSH
Related in: MedlinePlus