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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 augments the permeability of HUVEC monolayers.(a). HUVEC were grown on fibronectin- or collagen I-coated PET filter inserts for 20 hours to ensure confluence and treated with cilengitide (10 µM), CGP77675 (2.5 µM) or a combination thereof. Permeability was measured using the tracer molecule FITC-dextran. Cilengitide increased HUVEC monolayer permeability on both matrices and CGP77675 only partially prevented this increase. Results represent the increase in permeability of treated cultures relative to untreated controls at t = 0 and is given in arbitrary fluorescence units (AU). (b) Crystal violet staining of control and treated filters at the end of the assay (240 minutes) revealed that cilengitide did not cause extensive detachment of HUVEC but induced the appearance of retraced, dendritic-like cells (arrows). (Triplicate filters/condition, n = 3).
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pone-0004449-g007: Cilengitide augments the permeability of HUVEC monolayers.(a). HUVEC were grown on fibronectin- or collagen I-coated PET filter inserts for 20 hours to ensure confluence and treated with cilengitide (10 µM), CGP77675 (2.5 µM) or a combination thereof. Permeability was measured using the tracer molecule FITC-dextran. Cilengitide increased HUVEC monolayer permeability on both matrices and CGP77675 only partially prevented this increase. Results represent the increase in permeability of treated cultures relative to untreated controls at t = 0 and is given in arbitrary fluorescence units (AU). (b) Crystal violet staining of control and treated filters at the end of the assay (240 minutes) revealed that cilengitide did not cause extensive detachment of HUVEC but induced the appearance of retraced, dendritic-like cells (arrows). (Triplicate filters/condition, n = 3).

Mentions: VE-cadherin-mediated cell-cell adhesion and integrin-mediated cell-matrix adhesion are essential for maintaining endothelial cell monolayer tightness [36], [37]. Based on the above observations, we set up to test whether cilengitide treatment increased permeability of confluent HUVEC. Addition of cilengitide (10 µM) to HUVEC cultured on fibronectin or collagen-coated filter inserts, resulted in a time-dependent increase in transendothelial permeability (Figure 7a). Microscopic examination of the filters at the end of the assay (240 minutes) revealed that cilengitide induced morphological changes to the cultures, in particular the appearance of dark (dense) dendritic-like cells, consistent with cells that retracted or detached from the substrate (Figure 7b, arrows). CGP77675 (2.5 µM) partially abolished cilengitide-induced increased permeability but was ineffective in preventing the appearance of retracted cells (Figure 7a and 7b). As expected treatment of HUVEC cultured on vitronectin-coated filters resulted in massive cell detachment and increased permeability, consistent with αVβ3/αVβ5-mediated adhesion to this substrate (data not shown).


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 augments the permeability of HUVEC monolayers.(a). HUVEC were grown on fibronectin- or collagen I-coated PET filter inserts for 20 hours to ensure confluence and treated with cilengitide (10 µM), CGP77675 (2.5 µM) or a combination thereof. Permeability was measured using the tracer molecule FITC-dextran. Cilengitide increased HUVEC monolayer permeability on both matrices and CGP77675 only partially prevented this increase. Results represent the increase in permeability of treated cultures relative to untreated controls at t = 0 and is given in arbitrary fluorescence units (AU). (b) Crystal violet staining of control and treated filters at the end of the assay (240 minutes) revealed that cilengitide did not cause extensive detachment of HUVEC but induced the appearance of retraced, dendritic-like cells (arrows). (Triplicate filters/condition, n = 3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2636874&req=5

pone-0004449-g007: Cilengitide augments the permeability of HUVEC monolayers.(a). HUVEC were grown on fibronectin- or collagen I-coated PET filter inserts for 20 hours to ensure confluence and treated with cilengitide (10 µM), CGP77675 (2.5 µM) or a combination thereof. Permeability was measured using the tracer molecule FITC-dextran. Cilengitide increased HUVEC monolayer permeability on both matrices and CGP77675 only partially prevented this increase. Results represent the increase in permeability of treated cultures relative to untreated controls at t = 0 and is given in arbitrary fluorescence units (AU). (b) Crystal violet staining of control and treated filters at the end of the assay (240 minutes) revealed that cilengitide did not cause extensive detachment of HUVEC but induced the appearance of retraced, dendritic-like cells (arrows). (Triplicate filters/condition, n = 3).
Mentions: VE-cadherin-mediated cell-cell adhesion and integrin-mediated cell-matrix adhesion are essential for maintaining endothelial cell monolayer tightness [36], [37]. Based on the above observations, we set up to test whether cilengitide treatment increased permeability of confluent HUVEC. Addition of cilengitide (10 µM) to HUVEC cultured on fibronectin or collagen-coated filter inserts, resulted in a time-dependent increase in transendothelial permeability (Figure 7a). Microscopic examination of the filters at the end of the assay (240 minutes) revealed that cilengitide induced morphological changes to the cultures, in particular the appearance of dark (dense) dendritic-like cells, consistent with cells that retracted or detached from the substrate (Figure 7b, arrows). CGP77675 (2.5 µM) partially abolished cilengitide-induced increased permeability but was ineffective in preventing the appearance of retracted cells (Figure 7a and 7b). As expected treatment of HUVEC cultured on vitronectin-coated filters resulted in massive cell detachment and increased permeability, consistent with αVβ3/αVβ5-mediated adhesion to this substrate (data not shown).

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