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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 causes loss of αVβ3 from focal adhesions and promotes appearance of αVβ3 patches at the cell edge.HUVEC were plated on coverslips coated with fibronectin or collagen I and were treated with 10 µM of cilengitide for 20 minutes. The localization of the αVβ3 or β1 integrin (green) and paxillin (red) were monitored by immunofluorescence staining. In HUVEC plated on fibronectin αVβ3 was present at focal adhesions, while β1 was present at fibrillar adhesions. Cilengitide, but not EMD 135981, caused loss of αVβ3 from focal adhesions and appearance of αVβ3-positive thin patches at the cell edge (arrows). β1 localization was not altered by cilengitide. A similar effect on αVβ3 (arrows) was observed on cells plated on collagen I, with the difference that focal adhesions were less abundant on this matrix. Optical magnification: 400×; Bar: 10 µm. (n = 5).
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pone-0004449-g001: Cilengitide causes loss of αVβ3 from focal adhesions and promotes appearance of αVβ3 patches at the cell edge.HUVEC were plated on coverslips coated with fibronectin or collagen I and were treated with 10 µM of cilengitide for 20 minutes. The localization of the αVβ3 or β1 integrin (green) and paxillin (red) were monitored by immunofluorescence staining. In HUVEC plated on fibronectin αVβ3 was present at focal adhesions, while β1 was present at fibrillar adhesions. Cilengitide, but not EMD 135981, caused loss of αVβ3 from focal adhesions and appearance of αVβ3-positive thin patches at the cell edge (arrows). β1 localization was not altered by cilengitide. A similar effect on αVβ3 (arrows) was observed on cells plated on collagen I, with the difference that focal adhesions were less abundant on this matrix. Optical magnification: 400×; Bar: 10 µm. (n = 5).

Mentions: Cilengitide efficiently inhibits αVβ3-mediated cell adhesion and induces detachment of endothelial cells cultured on αVβ3 ligands, such as vitronectin or gelatin [13]. To test the effect of cilengitide on endothelial cells expressing αVβ3 but mostly using β1 integrins for their adhesion, we seeded HUVEC on fibronectin and collagen I. HUVEC use predominantly α5β1 to adhere to fibronectin (with minor contribution of αVβ3) and α1β1/α2β1 to adhere to collagen I [23]. Subsequently we exposed adherent HUVEC to cilengitide at a clinically-relevant concentration (i.e. 10 µM, [17]) or EMD135981, an Arg-Ala-Asp (RAD)-based inactive cyclopeptide. First we monitored the effect of cilengitide on αVβ3 localization. In HUVEC plated on fibronectin, αVβ3 was present at focal adhesions while β1 integrins clustered at fibrillar adhesions, as previously observed [24]. Cilengitide, but not EMD135981, caused loss of αVβ3 from paxillin-positive focal adhesions and promoted the appearance of thin, αVβ3-positive and paxillin negative linings at the cell edge (Figure 1, arrows). The localization of β1 integrins at fibrillar adhesions was not perturbed by cilengitide. HUVEC cultured on collagen I showed fewer focal adhesions while had well-developed fibrillar adhesions. Cilengitide treatment induced αVβ3 accumulation at the cell border without affecting β1 integrin localization (Figure 1).


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 causes loss of αVβ3 from focal adhesions and promotes appearance of αVβ3 patches at the cell edge.HUVEC were plated on coverslips coated with fibronectin or collagen I and were treated with 10 µM of cilengitide for 20 minutes. The localization of the αVβ3 or β1 integrin (green) and paxillin (red) were monitored by immunofluorescence staining. In HUVEC plated on fibronectin αVβ3 was present at focal adhesions, while β1 was present at fibrillar adhesions. Cilengitide, but not EMD 135981, caused loss of αVβ3 from focal adhesions and appearance of αVβ3-positive thin patches at the cell edge (arrows). β1 localization was not altered by cilengitide. A similar effect on αVβ3 (arrows) was observed on cells plated on collagen I, with the difference that focal adhesions were less abundant on this matrix. Optical magnification: 400×; Bar: 10 µm. (n = 5).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2636874&req=5

pone-0004449-g001: Cilengitide causes loss of αVβ3 from focal adhesions and promotes appearance of αVβ3 patches at the cell edge.HUVEC were plated on coverslips coated with fibronectin or collagen I and were treated with 10 µM of cilengitide for 20 minutes. The localization of the αVβ3 or β1 integrin (green) and paxillin (red) were monitored by immunofluorescence staining. In HUVEC plated on fibronectin αVβ3 was present at focal adhesions, while β1 was present at fibrillar adhesions. Cilengitide, but not EMD 135981, caused loss of αVβ3 from focal adhesions and appearance of αVβ3-positive thin patches at the cell edge (arrows). β1 localization was not altered by cilengitide. A similar effect on αVβ3 (arrows) was observed on cells plated on collagen I, with the difference that focal adhesions were less abundant on this matrix. Optical magnification: 400×; Bar: 10 µm. (n = 5).
Mentions: Cilengitide efficiently inhibits αVβ3-mediated cell adhesion and induces detachment of endothelial cells cultured on αVβ3 ligands, such as vitronectin or gelatin [13]. To test the effect of cilengitide on endothelial cells expressing αVβ3 but mostly using β1 integrins for their adhesion, we seeded HUVEC on fibronectin and collagen I. HUVEC use predominantly α5β1 to adhere to fibronectin (with minor contribution of αVβ3) and α1β1/α2β1 to adhere to collagen I [23]. Subsequently we exposed adherent HUVEC to cilengitide at a clinically-relevant concentration (i.e. 10 µM, [17]) or EMD135981, an Arg-Ala-Asp (RAD)-based inactive cyclopeptide. First we monitored the effect of cilengitide on αVβ3 localization. In HUVEC plated on fibronectin, αVβ3 was present at focal adhesions while β1 integrins clustered at fibrillar adhesions, as previously observed [24]. Cilengitide, but not EMD135981, caused loss of αVβ3 from paxillin-positive focal adhesions and promoted the appearance of thin, αVβ3-positive and paxillin negative linings at the cell edge (Figure 1, arrows). The localization of β1 integrins at fibrillar adhesions was not perturbed by cilengitide. HUVEC cultured on collagen I showed fewer focal adhesions while had well-developed fibrillar adhesions. Cilengitide treatment induced αVβ3 accumulation at the cell border without affecting β1 integrin localization (Figure 1).

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