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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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Related in: MedlinePlus

Adult MABs were injected into the right femoral artery of Sgca- and Sgca-/ JAM-A- mice. After 6 h, the hind limb muscles (gastrocnemius, tibialis anterior and quadriceps) were collected and the presence of migrated cells was quantified using qRT-PCR with nLacZ primers. The nLacZ RNA relative level (normalized to GAPDH expression) obtained for controls was set to 1, and the ratios for Sgca-/ JAM-A- ( n = 8) versus control mice ( Sgca-, n = 6) is shown. Fold increase has been extrapolated by data shown in Figure S1F.Sgca- mice were treated with a JAM-A neutralizing antibody (BV11, 3 mg/kg) or with non-related IgG (IgG, 3 mg/kg) as control. After 1 h, the mice were intra-arterially transplanted with adult MABs and 6 h later the muscles were collected and processed as described in A. nLacZ RNA relative levels obtained for the controls was set to 1, and the ratio for BV11 versus control (IgG) is shown. Fold increase has been extrapolated by data shown in Figure S1G.BV11 ( n = 3) or IgG ( n = 3) were given to Sgca-/scid/beige mice as described in B. Three weeks after the C57-nLacZ intra-arterial transplantations, the muscles were analyzed and representative cryosections of the gastrocnemius muscle stained for eosin and X-gal are shown. Scale bar: 40 μm.Quantification of the number of X-gal+ MABs in gastrocnemius cryosections. Scale bar: 40 μm. *** P < 0.001.Immunofluorescence staining of Sgca (red), β-galactosidase (β-gal, green), Hoechst (blue) and laminin (white, lower images of both panels) were performed on sections adjacent to those shown in C (transplanted with MABs) and on contralateral not transplanted muscles (w/o MABs) as a control. Merged images of red, green and blue signals are shown (upper images of both panels). Scale bar: 80 μm.Quantification of Sgca+ fibers shown in E, upper panel (transplanted with MABs). *** P < 0.0001. Data are expressed as means ± s.e.m.
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fig02: Adult MABs were injected into the right femoral artery of Sgca- and Sgca-/ JAM-A- mice. After 6 h, the hind limb muscles (gastrocnemius, tibialis anterior and quadriceps) were collected and the presence of migrated cells was quantified using qRT-PCR with nLacZ primers. The nLacZ RNA relative level (normalized to GAPDH expression) obtained for controls was set to 1, and the ratios for Sgca-/ JAM-A- ( n = 8) versus control mice ( Sgca-, n = 6) is shown. Fold increase has been extrapolated by data shown in Figure S1F.Sgca- mice were treated with a JAM-A neutralizing antibody (BV11, 3 mg/kg) or with non-related IgG (IgG, 3 mg/kg) as control. After 1 h, the mice were intra-arterially transplanted with adult MABs and 6 h later the muscles were collected and processed as described in A. nLacZ RNA relative levels obtained for the controls was set to 1, and the ratio for BV11 versus control (IgG) is shown. Fold increase has been extrapolated by data shown in Figure S1G.BV11 ( n = 3) or IgG ( n = 3) were given to Sgca-/scid/beige mice as described in B. Three weeks after the C57-nLacZ intra-arterial transplantations, the muscles were analyzed and representative cryosections of the gastrocnemius muscle stained for eosin and X-gal are shown. Scale bar: 40 μm.Quantification of the number of X-gal+ MABs in gastrocnemius cryosections. Scale bar: 40 μm. *** P < 0.001.Immunofluorescence staining of Sgca (red), β-galactosidase (β-gal, green), Hoechst (blue) and laminin (white, lower images of both panels) were performed on sections adjacent to those shown in C (transplanted with MABs) and on contralateral not transplanted muscles (w/o MABs) as a control. Merged images of red, green and blue signals are shown (upper images of both panels). Scale bar: 80 μm.Quantification of Sgca+ fibers shown in E, upper panel (transplanted with MABs). *** P < 0.0001. Data are expressed as means ± s.e.m.

Mentions: The in vivo migration of MABs to the muscle tissue was then assessed in these Sgca- and Sgca-/ JAM-A- mice, using adult murine MABs. Indeed, intra-arterially (femoral artery) transplanted MABs migrated significantly more efficiently to the muscles of the Sgca-/ JAM-A- mice than to those of the Sgca- control mice (Fig 2A and supplementary Fig S1F).


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)

Adult MABs were injected into the right femoral artery of Sgca- and Sgca-/ JAM-A- mice. After 6 h, the hind limb muscles (gastrocnemius, tibialis anterior and quadriceps) were collected and the presence of migrated cells was quantified using qRT-PCR with nLacZ primers. The nLacZ RNA relative level (normalized to GAPDH expression) obtained for controls was set to 1, and the ratios for Sgca-/ JAM-A- ( n = 8) versus control mice ( Sgca-, n = 6) is shown. Fold increase has been extrapolated by data shown in Figure S1F.Sgca- mice were treated with a JAM-A neutralizing antibody (BV11, 3 mg/kg) or with non-related IgG (IgG, 3 mg/kg) as control. After 1 h, the mice were intra-arterially transplanted with adult MABs and 6 h later the muscles were collected and processed as described in A. nLacZ RNA relative levels obtained for the controls was set to 1, and the ratio for BV11 versus control (IgG) is shown. Fold increase has been extrapolated by data shown in Figure S1G.BV11 ( n = 3) or IgG ( n = 3) were given to Sgca-/scid/beige mice as described in B. Three weeks after the C57-nLacZ intra-arterial transplantations, the muscles were analyzed and representative cryosections of the gastrocnemius muscle stained for eosin and X-gal are shown. Scale bar: 40 μm.Quantification of the number of X-gal+ MABs in gastrocnemius cryosections. Scale bar: 40 μm. *** P < 0.001.Immunofluorescence staining of Sgca (red), β-galactosidase (β-gal, green), Hoechst (blue) and laminin (white, lower images of both panels) were performed on sections adjacent to those shown in C (transplanted with MABs) and on contralateral not transplanted muscles (w/o MABs) as a control. Merged images of red, green and blue signals are shown (upper images of both panels). Scale bar: 80 μm.Quantification of Sgca+ fibers shown in E, upper panel (transplanted with MABs). *** P < 0.0001. Data are expressed as means ± s.e.m.
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Related In: Results  -  Collection

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fig02: Adult MABs were injected into the right femoral artery of Sgca- and Sgca-/ JAM-A- mice. After 6 h, the hind limb muscles (gastrocnemius, tibialis anterior and quadriceps) were collected and the presence of migrated cells was quantified using qRT-PCR with nLacZ primers. The nLacZ RNA relative level (normalized to GAPDH expression) obtained for controls was set to 1, and the ratios for Sgca-/ JAM-A- ( n = 8) versus control mice ( Sgca-, n = 6) is shown. Fold increase has been extrapolated by data shown in Figure S1F.Sgca- mice were treated with a JAM-A neutralizing antibody (BV11, 3 mg/kg) or with non-related IgG (IgG, 3 mg/kg) as control. After 1 h, the mice were intra-arterially transplanted with adult MABs and 6 h later the muscles were collected and processed as described in A. nLacZ RNA relative levels obtained for the controls was set to 1, and the ratio for BV11 versus control (IgG) is shown. Fold increase has been extrapolated by data shown in Figure S1G.BV11 ( n = 3) or IgG ( n = 3) were given to Sgca-/scid/beige mice as described in B. Three weeks after the C57-nLacZ intra-arterial transplantations, the muscles were analyzed and representative cryosections of the gastrocnemius muscle stained for eosin and X-gal are shown. Scale bar: 40 μm.Quantification of the number of X-gal+ MABs in gastrocnemius cryosections. Scale bar: 40 μm. *** P < 0.001.Immunofluorescence staining of Sgca (red), β-galactosidase (β-gal, green), Hoechst (blue) and laminin (white, lower images of both panels) were performed on sections adjacent to those shown in C (transplanted with MABs) and on contralateral not transplanted muscles (w/o MABs) as a control. Merged images of red, green and blue signals are shown (upper images of both panels). Scale bar: 80 μm.Quantification of Sgca+ fibers shown in E, upper panel (transplanted with MABs). *** P < 0.0001. Data are expressed as means ± s.e.m.
Mentions: The in vivo migration of MABs to the muscle tissue was then assessed in these Sgca- and Sgca-/ JAM-A- mice, using adult murine MABs. Indeed, intra-arterially (femoral artery) transplanted MABs migrated significantly more efficiently to the muscles of the Sgca-/ JAM-A- mice than to those of the Sgca- control mice (Fig 2A and supplementary Fig S1F).

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