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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

D16-GFP embryonic or C57-nLacZ adult murine mesoangioblasts (MABs) were injected into the right femoral artery of WT, PECAM-1- and JAM-A- age-matched mice (treated with cardiotoxin 24 h before transplantation) as indicated. 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 GFP or nLacZ primers. The RNA levels were normalized using GAPDH. The RNA relative levels for the controls were set to 1, and the ratios for PECAM-1- ( n = 7) or JAM-A- ( n = 10) versus WT ( n = 17) are shown for embryonic (left) and adult (right) murine MABs. Fold increases have been extrapolated by data shown in Figure S1A–E.Representative Hematoxilin and Eosin (H&E) staining of JAM-A-WT and JAM-A- mice from A after cardiotoxin treatment. Scale bars: 50 μm.Representative cryosections of the gastrocnemius muscle of JAM-A-WT and JAM-A- mice stained for with H&E and X-gal. Asterisks indicate donor cells. Scale bars: 50 μm.Quantification of X-gal+ MABs in cryosections illustrated in C. ** P < 0.05. Data are expressed as means ± s.e.m.
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fig01: D16-GFP embryonic or C57-nLacZ adult murine mesoangioblasts (MABs) were injected into the right femoral artery of WT, PECAM-1- and JAM-A- age-matched mice (treated with cardiotoxin 24 h before transplantation) as indicated. 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 GFP or nLacZ primers. The RNA levels were normalized using GAPDH. The RNA relative levels for the controls were set to 1, and the ratios for PECAM-1- ( n = 7) or JAM-A- ( n = 10) versus WT ( n = 17) are shown for embryonic (left) and adult (right) murine MABs. Fold increases have been extrapolated by data shown in Figure S1A–E.Representative Hematoxilin and Eosin (H&E) staining of JAM-A-WT and JAM-A- mice from A after cardiotoxin treatment. Scale bars: 50 μm.Representative cryosections of the gastrocnemius muscle of JAM-A-WT and JAM-A- mice stained for with H&E and X-gal. Asterisks indicate donor cells. Scale bars: 50 μm.Quantification of X-gal+ MABs in cryosections illustrated in C. ** P < 0.05. Data are expressed as means ± s.e.m.

Mentions: To investigate the involvement of the endothelium in MAB engraftment, we focused on two endothelial junction proteins with established functions in leukocyte diapedesis: JAM-A and PECAM-1 (Bazzoni & Dejana, 2004; Woodfin et al, 2007). At first, the in vivo migration of MABs from the vessel lumen to the muscle interstitial tissues was assessed in genetically modified JAM-A and PECAM-1 deficient mice ( JAM-A- and PECAM-1- mice, respectively) (Duncan et al, 1999; Cera et al, 2004). To this end, green fluorescent protein (GFP)-expressing murine embryonic MABs (D16-GFP) (Sampaolesi et al, 2003) were injected into the right femoral artery of wild-type (WT), JAM-A- and PECAM-1- age-matched mice after acute muscular injury caused by a previous intra-muscular injection of cardiotoxin. After 6 h, the mice were sacrificed and the hind limb muscles (gastrocnemius, tibialis anterior and quadriceps) were collected and their RNA was extracted. qRT-PCR of GFP expression was used to evaluate the presence of migrated cells. Embryonic MABs migrated significantly more efficiently to the injured muscles of the JAM-A- mice than WT, although they did not show a significant increase in engraftment over WT when injected into the PECAM-1- mice (Fig 1A, on the left of the dashed line and supplementary Fig S1A–D). This suggested a selective and specific role for JAM-A in MAB transmigration across the endothelium.


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)

D16-GFP embryonic or C57-nLacZ adult murine mesoangioblasts (MABs) were injected into the right femoral artery of WT, PECAM-1- and JAM-A- age-matched mice (treated with cardiotoxin 24 h before transplantation) as indicated. 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 GFP or nLacZ primers. The RNA levels were normalized using GAPDH. The RNA relative levels for the controls were set to 1, and the ratios for PECAM-1- ( n = 7) or JAM-A- ( n = 10) versus WT ( n = 17) are shown for embryonic (left) and adult (right) murine MABs. Fold increases have been extrapolated by data shown in Figure S1A–E.Representative Hematoxilin and Eosin (H&E) staining of JAM-A-WT and JAM-A- mice from A after cardiotoxin treatment. Scale bars: 50 μm.Representative cryosections of the gastrocnemius muscle of JAM-A-WT and JAM-A- mice stained for with H&E and X-gal. Asterisks indicate donor cells. Scale bars: 50 μm.Quantification of X-gal+ MABs in cryosections illustrated in C. ** P < 0.05. Data are expressed as means ± s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig01: D16-GFP embryonic or C57-nLacZ adult murine mesoangioblasts (MABs) were injected into the right femoral artery of WT, PECAM-1- and JAM-A- age-matched mice (treated with cardiotoxin 24 h before transplantation) as indicated. 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 GFP or nLacZ primers. The RNA levels were normalized using GAPDH. The RNA relative levels for the controls were set to 1, and the ratios for PECAM-1- ( n = 7) or JAM-A- ( n = 10) versus WT ( n = 17) are shown for embryonic (left) and adult (right) murine MABs. Fold increases have been extrapolated by data shown in Figure S1A–E.Representative Hematoxilin and Eosin (H&E) staining of JAM-A-WT and JAM-A- mice from A after cardiotoxin treatment. Scale bars: 50 μm.Representative cryosections of the gastrocnemius muscle of JAM-A-WT and JAM-A- mice stained for with H&E and X-gal. Asterisks indicate donor cells. Scale bars: 50 μm.Quantification of X-gal+ MABs in cryosections illustrated in C. ** P < 0.05. Data are expressed as means ± s.e.m.
Mentions: To investigate the involvement of the endothelium in MAB engraftment, we focused on two endothelial junction proteins with established functions in leukocyte diapedesis: JAM-A and PECAM-1 (Bazzoni & Dejana, 2004; Woodfin et al, 2007). At first, the in vivo migration of MABs from the vessel lumen to the muscle interstitial tissues was assessed in genetically modified JAM-A and PECAM-1 deficient mice ( JAM-A- and PECAM-1- mice, respectively) (Duncan et al, 1999; Cera et al, 2004). To this end, green fluorescent protein (GFP)-expressing murine embryonic MABs (D16-GFP) (Sampaolesi et al, 2003) were injected into the right femoral artery of wild-type (WT), JAM-A- and PECAM-1- age-matched mice after acute muscular injury caused by a previous intra-muscular injection of cardiotoxin. After 6 h, the mice were sacrificed and the hind limb muscles (gastrocnemius, tibialis anterior and quadriceps) were collected and their RNA was extracted. qRT-PCR of GFP expression was used to evaluate the presence of migrated cells. Embryonic MABs migrated significantly more efficiently to the injured muscles of the JAM-A- mice than WT, although they did not show a significant increase in engraftment over WT when injected into the PECAM-1- mice (Fig 1A, on the left of the dashed line and supplementary Fig S1A–D). This suggested a selective and specific role for JAM-A in MAB transmigration across the endothelium.

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