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Targeting endothelial junctional adhesion molecule-A/ EPAC/ Rap-1 axis as a novel strategy to increase stem cell engraftment in dystrophic muscles.

Giannotta M, Benedetti S, Tedesco FS, Corada M, Trani M, D'Antuono R, Millet Q, Orsenigo F, Gálvez BG, Cossu G, Dejana E - EMBO Mol Med (2013)

Bottom Line: Experimental clinical treatments include intra-arterial administration of vessel-associated stem cells, called mesoangioblasts (MABs).As a consequence, junction tightening is reduced, allowing MAB diapedesis.Notably, pharmacological inhibition of Rap-1 increases MAB engraftment in dystrophic muscle, which results into a significant improvement of muscle function offering a novel strategy for stem cell-based therapies.

View Article: PubMed Central - PubMed

Affiliation: FIRC Institute of Molecular Oncology Foundation (IFOM), Milan, Italy.

ABSTRACT
Muscular dystrophies are severe genetic diseases for which no efficacious therapies exist. Experimental clinical treatments include intra-arterial administration of vessel-associated stem cells, called mesoangioblasts (MABs). However, one of the limitations of this approach is the relatively low number of cells that engraft the diseased tissue, due, at least in part, to the sub-optimal efficiency of extravasation, whose mechanisms for MAB are unknown. Leukocytes emigrate into the inflamed tissues by crossing endothelial cell-to-cell junctions and junctional proteins direct and control leukocyte diapedesis. Here, we identify the endothelial junctional protein JAM-A as a key regulator of MAB extravasation. We show that JAM-A gene inactivation and JAM-A blocking antibodies strongly enhance MAB engraftment in dystrophic muscle. In the absence of JAM-A, the exchange factors EPAC-1 and 2 are down-regulated, which prevents the activation of the small GTPase Rap-1. As a consequence, junction tightening is reduced, allowing MAB diapedesis. Notably, pharmacological inhibition of Rap-1 increases MAB engraftment in dystrophic muscle, which results into a significant improvement of muscle function offering a novel strategy for stem cell-based therapies.

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Endothelial cells isolated from JAM-A-WT (left) and JAM-A- (right) mice were seeded onto glutaraldehyde-crosslinked gelatin-coated filters (Transwell). 6-carboxyfluorescin diacetate (6-CFDA)-labeled embryonic (top) and adult (bottom) MABs were added to the upper chamber and allowed to migrate for 6 h. The migrated MABs (green) on the lower sides of the filters were fixed and counted. A representative experiment of five independent experiments, each in triplicate, is shown. Scale bar: 100 μm.Quantification of the number of migrated MABs per area, as illustrated in A. ** P < 0.001. Data are means ± s.e.m. from five independent experiments, each in triplicate.As in A, except that JAM-A-WT endothelial cells were pre-treated (3 h) and further incubated (6 h) with vehicle, non-related IgG and BV11 (20 μg/ml) (left and middle, respectively). JAM-A- endothelial cells were also incubated with vehicle (right) as a further internal control, illustrating that increased MAB transmigration due to acute inhibition of JAM-A is comparable with their capacity to migrate in the absence of JAM-A. Representative experiment of four independent experiments carried out in triplicate. Scale bars: 100 μm.Quantification of migrated embryonic MABs through JAM-A-WT (white and black bars) and JAM-A- (grey bar) endothelial cells per area, as illustrated in C. ** P < 0.001. Data are means ± s.e.m. from four independent experiments, each in triplicate.6-CFDA-labelled embryonic (left) and adult (right) MABs were incubated with non-related IgGs (white bars), BV11 (20 μg/ml), (black bars) for 3 h. Then the MABs were seeded on filters and allowed to migrate for a further 3 h in the presence of the indicated agents. The migrated MABs on the lower side of the filters (green) were fixed and counted. Quantification of migrated cells per area is shown. Data are means ± s.e.m. from three independent experiments, each in triplicate.
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fig03: Endothelial cells isolated from JAM-A-WT (left) and JAM-A- (right) mice were seeded onto glutaraldehyde-crosslinked gelatin-coated filters (Transwell). 6-carboxyfluorescin diacetate (6-CFDA)-labeled embryonic (top) and adult (bottom) MABs were added to the upper chamber and allowed to migrate for 6 h. The migrated MABs (green) on the lower sides of the filters were fixed and counted. A representative experiment of five independent experiments, each in triplicate, is shown. Scale bar: 100 μm.Quantification of the number of migrated MABs per area, as illustrated in A. ** P < 0.001. Data are means ± s.e.m. from five independent experiments, each in triplicate.As in A, except that JAM-A-WT endothelial cells were pre-treated (3 h) and further incubated (6 h) with vehicle, non-related IgG and BV11 (20 μg/ml) (left and middle, respectively). JAM-A- endothelial cells were also incubated with vehicle (right) as a further internal control, illustrating that increased MAB transmigration due to acute inhibition of JAM-A is comparable with their capacity to migrate in the absence of JAM-A. Representative experiment of four independent experiments carried out in triplicate. Scale bars: 100 μm.Quantification of migrated embryonic MABs through JAM-A-WT (white and black bars) and JAM-A- (grey bar) endothelial cells per area, as illustrated in C. ** P < 0.001. Data are means ± s.e.m. from four independent experiments, each in triplicate.6-CFDA-labelled embryonic (left) and adult (right) MABs were incubated with non-related IgGs (white bars), BV11 (20 μg/ml), (black bars) for 3 h. Then the MABs were seeded on filters and allowed to migrate for a further 3 h in the presence of the indicated agents. The migrated MABs on the lower side of the filters (green) were fixed and counted. Quantification of migrated cells per area is shown. Data are means ± s.e.m. from three independent experiments, each in triplicate.

Mentions: We used endothelial cells isolated from the lungs of WT and JAM-A- mice (Cera et al, 2004). These cells were grown as confluent monolayers on glutaraldehyde-cross-linked gelatin-coated filters. We then tested embryonic and adult murine MAB transmigration through these endothelial monolayers. Similar to what observed in vivo, the number of MABs that crossed the endothelium was significantly increased in the absence of JAM-A (Fig 3A and B). Furthermore, pre-incubation of the murine endothelial cells with BV11 strongly enhanced the transmigration of embryonic murine MABs (Fig 3C, left panel), with a 6-7-fold increase compared to the number of MABs that crossed the endothelium in the presence of vehicle or non-related IgG (Fig 3D). A similar increase in MAB transmigration was also achieved using monolayers of JAM-A- endothelial cells, suggesting that chronic and acute JAM-A inhibition results in similar effects (Fig 3C and D).


Targeting endothelial junctional adhesion molecule-A/ EPAC/ Rap-1 axis as a novel strategy to increase stem cell engraftment in dystrophic muscles.

Giannotta M, Benedetti S, Tedesco FS, Corada M, Trani M, D'Antuono R, Millet Q, Orsenigo F, Gálvez BG, Cossu G, Dejana E - EMBO Mol Med (2013)

Endothelial cells isolated from JAM-A-WT (left) and JAM-A- (right) mice were seeded onto glutaraldehyde-crosslinked gelatin-coated filters (Transwell). 6-carboxyfluorescin diacetate (6-CFDA)-labeled embryonic (top) and adult (bottom) MABs were added to the upper chamber and allowed to migrate for 6 h. The migrated MABs (green) on the lower sides of the filters were fixed and counted. A representative experiment of five independent experiments, each in triplicate, is shown. Scale bar: 100 μm.Quantification of the number of migrated MABs per area, as illustrated in A. ** P < 0.001. Data are means ± s.e.m. from five independent experiments, each in triplicate.As in A, except that JAM-A-WT endothelial cells were pre-treated (3 h) and further incubated (6 h) with vehicle, non-related IgG and BV11 (20 μg/ml) (left and middle, respectively). JAM-A- endothelial cells were also incubated with vehicle (right) as a further internal control, illustrating that increased MAB transmigration due to acute inhibition of JAM-A is comparable with their capacity to migrate in the absence of JAM-A. Representative experiment of four independent experiments carried out in triplicate. Scale bars: 100 μm.Quantification of migrated embryonic MABs through JAM-A-WT (white and black bars) and JAM-A- (grey bar) endothelial cells per area, as illustrated in C. ** P < 0.001. Data are means ± s.e.m. from four independent experiments, each in triplicate.6-CFDA-labelled embryonic (left) and adult (right) MABs were incubated with non-related IgGs (white bars), BV11 (20 μg/ml), (black bars) for 3 h. Then the MABs were seeded on filters and allowed to migrate for a further 3 h in the presence of the indicated agents. The migrated MABs on the lower side of the filters (green) were fixed and counted. Quantification of migrated cells per area is shown. Data are means ± s.e.m. from three independent experiments, each in triplicate.
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Related In: Results  -  Collection

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fig03: Endothelial cells isolated from JAM-A-WT (left) and JAM-A- (right) mice were seeded onto glutaraldehyde-crosslinked gelatin-coated filters (Transwell). 6-carboxyfluorescin diacetate (6-CFDA)-labeled embryonic (top) and adult (bottom) MABs were added to the upper chamber and allowed to migrate for 6 h. The migrated MABs (green) on the lower sides of the filters were fixed and counted. A representative experiment of five independent experiments, each in triplicate, is shown. Scale bar: 100 μm.Quantification of the number of migrated MABs per area, as illustrated in A. ** P < 0.001. Data are means ± s.e.m. from five independent experiments, each in triplicate.As in A, except that JAM-A-WT endothelial cells were pre-treated (3 h) and further incubated (6 h) with vehicle, non-related IgG and BV11 (20 μg/ml) (left and middle, respectively). JAM-A- endothelial cells were also incubated with vehicle (right) as a further internal control, illustrating that increased MAB transmigration due to acute inhibition of JAM-A is comparable with their capacity to migrate in the absence of JAM-A. Representative experiment of four independent experiments carried out in triplicate. Scale bars: 100 μm.Quantification of migrated embryonic MABs through JAM-A-WT (white and black bars) and JAM-A- (grey bar) endothelial cells per area, as illustrated in C. ** P < 0.001. Data are means ± s.e.m. from four independent experiments, each in triplicate.6-CFDA-labelled embryonic (left) and adult (right) MABs were incubated with non-related IgGs (white bars), BV11 (20 μg/ml), (black bars) for 3 h. Then the MABs were seeded on filters and allowed to migrate for a further 3 h in the presence of the indicated agents. The migrated MABs on the lower side of the filters (green) were fixed and counted. Quantification of migrated cells per area is shown. Data are means ± s.e.m. from three independent experiments, each in triplicate.
Mentions: We used endothelial cells isolated from the lungs of WT and JAM-A- mice (Cera et al, 2004). These cells were grown as confluent monolayers on glutaraldehyde-cross-linked gelatin-coated filters. We then tested embryonic and adult murine MAB transmigration through these endothelial monolayers. Similar to what observed in vivo, the number of MABs that crossed the endothelium was significantly increased in the absence of JAM-A (Fig 3A and B). Furthermore, pre-incubation of the murine endothelial cells with BV11 strongly enhanced the transmigration of embryonic murine MABs (Fig 3C, left panel), with a 6-7-fold increase compared to the number of MABs that crossed the endothelium in the presence of vehicle or non-related IgG (Fig 3D). A similar increase in MAB transmigration was also achieved using monolayers of JAM-A- endothelial cells, suggesting that chronic and acute JAM-A inhibition results in similar effects (Fig 3C and D).

Bottom Line: Experimental clinical treatments include intra-arterial administration of vessel-associated stem cells, called mesoangioblasts (MABs).As a consequence, junction tightening is reduced, allowing MAB diapedesis.Notably, pharmacological inhibition of Rap-1 increases MAB engraftment in dystrophic muscle, which results into a significant improvement of muscle function offering a novel strategy for stem cell-based therapies.

View Article: PubMed Central - PubMed

Affiliation: FIRC Institute of Molecular Oncology Foundation (IFOM), Milan, Italy.

ABSTRACT
Muscular dystrophies are severe genetic diseases for which no efficacious therapies exist. Experimental clinical treatments include intra-arterial administration of vessel-associated stem cells, called mesoangioblasts (MABs). However, one of the limitations of this approach is the relatively low number of cells that engraft the diseased tissue, due, at least in part, to the sub-optimal efficiency of extravasation, whose mechanisms for MAB are unknown. Leukocytes emigrate into the inflamed tissues by crossing endothelial cell-to-cell junctions and junctional proteins direct and control leukocyte diapedesis. Here, we identify the endothelial junctional protein JAM-A as a key regulator of MAB extravasation. We show that JAM-A gene inactivation and JAM-A blocking antibodies strongly enhance MAB engraftment in dystrophic muscle. In the absence of JAM-A, the exchange factors EPAC-1 and 2 are down-regulated, which prevents the activation of the small GTPase Rap-1. As a consequence, junction tightening is reduced, allowing MAB diapedesis. Notably, pharmacological inhibition of Rap-1 increases MAB engraftment in dystrophic muscle, which results into a significant improvement of muscle function offering a novel strategy for stem cell-based therapies.

Show MeSH
Related in: MedlinePlus