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Overexpression of CD44 in neural precursor cells improves trans-endothelial migration and facilitates their invasion of perivascular tissues in vivo.

Deboux C, Ladraa S, Cazaubon S, Ghribi-Mallah S, Weiss N, Chaverot N, Couraud PO, Baron-Van Evercooren A - PLoS ONE (2013)

Bottom Line: In view of the role of CD44 in NPCs trans-endothelial migration in vitro, we questioned presently the benefit of CD44 overexpression by NPCs in vitro and in vivo, in EAE mice.Moreover, CD44 overexpression by NPCs improved significantly their elongation, spreading and number of filopodia over the extracellular matrix protein laminin in vitro.We then tested the effect of CD44 overexpression after i.v. delivery in the tail vein of EAE mice.

View Article: PubMed Central - PubMed

Affiliation: Université Pierre et Marie Curie-Paris 6, Centre de Recherche de l'Institut du Cerveau et de la Moelle Epinière, UMR-S975, Paris, France.

ABSTRACT
Neural precursor (NPC) based therapies are used to restore neurons or oligodendrocytes and/or provide neuroprotection in a large variety of neurological diseases. In multiple sclerosis models, intravenously (i.v) -delivered NPCs reduced clinical signs via immunomodulation. We demonstrated recently that NPCs were able to cross cerebral endothelial cells in vitro and that the multifunctional signalling molecule, CD44 involved in trans-endothelial migration of lymphocytes to sites of inflammation, plays a crucial role in extravasation of syngeneic NPCs. In view of the role of CD44 in NPCs trans-endothelial migration in vitro, we questioned presently the benefit of CD44 overexpression by NPCs in vitro and in vivo, in EAE mice. We show that overexpression of CD44 by NPCs enhanced over 2 folds their trans-endothelial migration in vitro, without impinging on the proliferation or differentiation potential of the transduced cells. Moreover, CD44 overexpression by NPCs improved significantly their elongation, spreading and number of filopodia over the extracellular matrix protein laminin in vitro. We then tested the effect of CD44 overexpression after i.v. delivery in the tail vein of EAE mice. CD44 overexpression was functional invivo as it accelerated trans-endothelial migration and facilitated invasion of HA expressing perivascular sites. These in vitro and in vivo data suggest that CD44 may be crucial not only for NPC crossing the endothelial layer but also for facilitating invasion of extravascular tissues.

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Effects of CD44 overexpression on trans-endothelial migration and spreading in vitro.(A, B) Trans-endothelial migration of actin eGFP CD44-NPCs (+) and control NPCs (-); quantification was performed using immunofluorescence for (A) eGFP or (B) c-myc. In response to SDF1α cells overexpressing CD44 migrate 2 fold more compared to control cells (experiments made in triplicate). (C, D) Spreading of NPCs in vitro on laminin: the number and length of filopodia are increased. (E, F) CD44 overexpressing cells are more elongated and spread than control NPCs. (* = p<0.5; ** = p<0.01, *** = p<0.01). (G) Immunocytochemistry for actin illustrating filopodia (arrowheads) in control-NPCs (Gi, Gii) and CD44-NPCs (Giii, Giv).
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pone-0057430-g003: Effects of CD44 overexpression on trans-endothelial migration and spreading in vitro.(A, B) Trans-endothelial migration of actin eGFP CD44-NPCs (+) and control NPCs (-); quantification was performed using immunofluorescence for (A) eGFP or (B) c-myc. In response to SDF1α cells overexpressing CD44 migrate 2 fold more compared to control cells (experiments made in triplicate). (C, D) Spreading of NPCs in vitro on laminin: the number and length of filopodia are increased. (E, F) CD44 overexpressing cells are more elongated and spread than control NPCs. (* = p<0.5; ** = p<0.01, *** = p<0.01). (G) Immunocytochemistry for actin illustrating filopodia (arrowheads) in control-NPCs (Gi, Gii) and CD44-NPCs (Giii, Giv).

Mentions: Our previous data showed that CD44 mediates trans-endothelial migration in vitro. Having established that CD44 overexpression by NPC does not affect their immature phenotype and their ability to differentiate into the major neural phenotypes, we analyzed whether CD44 overexpression would favour trans-endothelial migration using the previously described bEnd3 mouse endothelial model (27). bEND endothelial cells were grown to confluence on collagen-cultured inserts. Migration of actin-eGFP CD44-NPCs or control actin-eGFP NPCs, was performed overnight under a gradient of SDF-1alpha, a known chemo attractant of lymphocytes, neutrophils and NPCs (19, 27). Migrated NPCs adhering to the lower surface of the transwell filters, were imaged by confocal microscopy and quantified. Data indicate that trans-endothelial migration of CD44-NPCs in response to SDF-1alpha was increased by 2.5 fold compared to control cells (Fig. 3A, B).


Overexpression of CD44 in neural precursor cells improves trans-endothelial migration and facilitates their invasion of perivascular tissues in vivo.

Deboux C, Ladraa S, Cazaubon S, Ghribi-Mallah S, Weiss N, Chaverot N, Couraud PO, Baron-Van Evercooren A - PLoS ONE (2013)

Effects of CD44 overexpression on trans-endothelial migration and spreading in vitro.(A, B) Trans-endothelial migration of actin eGFP CD44-NPCs (+) and control NPCs (-); quantification was performed using immunofluorescence for (A) eGFP or (B) c-myc. In response to SDF1α cells overexpressing CD44 migrate 2 fold more compared to control cells (experiments made in triplicate). (C, D) Spreading of NPCs in vitro on laminin: the number and length of filopodia are increased. (E, F) CD44 overexpressing cells are more elongated and spread than control NPCs. (* = p<0.5; ** = p<0.01, *** = p<0.01). (G) Immunocytochemistry for actin illustrating filopodia (arrowheads) in control-NPCs (Gi, Gii) and CD44-NPCs (Giii, Giv).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585392&req=5

pone-0057430-g003: Effects of CD44 overexpression on trans-endothelial migration and spreading in vitro.(A, B) Trans-endothelial migration of actin eGFP CD44-NPCs (+) and control NPCs (-); quantification was performed using immunofluorescence for (A) eGFP or (B) c-myc. In response to SDF1α cells overexpressing CD44 migrate 2 fold more compared to control cells (experiments made in triplicate). (C, D) Spreading of NPCs in vitro on laminin: the number and length of filopodia are increased. (E, F) CD44 overexpressing cells are more elongated and spread than control NPCs. (* = p<0.5; ** = p<0.01, *** = p<0.01). (G) Immunocytochemistry for actin illustrating filopodia (arrowheads) in control-NPCs (Gi, Gii) and CD44-NPCs (Giii, Giv).
Mentions: Our previous data showed that CD44 mediates trans-endothelial migration in vitro. Having established that CD44 overexpression by NPC does not affect their immature phenotype and their ability to differentiate into the major neural phenotypes, we analyzed whether CD44 overexpression would favour trans-endothelial migration using the previously described bEnd3 mouse endothelial model (27). bEND endothelial cells were grown to confluence on collagen-cultured inserts. Migration of actin-eGFP CD44-NPCs or control actin-eGFP NPCs, was performed overnight under a gradient of SDF-1alpha, a known chemo attractant of lymphocytes, neutrophils and NPCs (19, 27). Migrated NPCs adhering to the lower surface of the transwell filters, were imaged by confocal microscopy and quantified. Data indicate that trans-endothelial migration of CD44-NPCs in response to SDF-1alpha was increased by 2.5 fold compared to control cells (Fig. 3A, B).

Bottom Line: In view of the role of CD44 in NPCs trans-endothelial migration in vitro, we questioned presently the benefit of CD44 overexpression by NPCs in vitro and in vivo, in EAE mice.Moreover, CD44 overexpression by NPCs improved significantly their elongation, spreading and number of filopodia over the extracellular matrix protein laminin in vitro.We then tested the effect of CD44 overexpression after i.v. delivery in the tail vein of EAE mice.

View Article: PubMed Central - PubMed

Affiliation: Université Pierre et Marie Curie-Paris 6, Centre de Recherche de l'Institut du Cerveau et de la Moelle Epinière, UMR-S975, Paris, France.

ABSTRACT
Neural precursor (NPC) based therapies are used to restore neurons or oligodendrocytes and/or provide neuroprotection in a large variety of neurological diseases. In multiple sclerosis models, intravenously (i.v) -delivered NPCs reduced clinical signs via immunomodulation. We demonstrated recently that NPCs were able to cross cerebral endothelial cells in vitro and that the multifunctional signalling molecule, CD44 involved in trans-endothelial migration of lymphocytes to sites of inflammation, plays a crucial role in extravasation of syngeneic NPCs. In view of the role of CD44 in NPCs trans-endothelial migration in vitro, we questioned presently the benefit of CD44 overexpression by NPCs in vitro and in vivo, in EAE mice. We show that overexpression of CD44 by NPCs enhanced over 2 folds their trans-endothelial migration in vitro, without impinging on the proliferation or differentiation potential of the transduced cells. Moreover, CD44 overexpression by NPCs improved significantly their elongation, spreading and number of filopodia over the extracellular matrix protein laminin in vitro. We then tested the effect of CD44 overexpression after i.v. delivery in the tail vein of EAE mice. CD44 overexpression was functional invivo as it accelerated trans-endothelial migration and facilitated invasion of HA expressing perivascular sites. These in vitro and in vivo data suggest that CD44 may be crucial not only for NPC crossing the endothelial layer but also for facilitating invasion of extravascular tissues.

Show MeSH
Related in: MedlinePlus