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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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HUVECs with stable scrambled shRNA (Ctrl) or JAM-A targeting shRNAs (#51, #49, #50, #52) were generated (see Materials and Methods) and then homogenized. The cell lysates were analyzed by immunoblotting for JAM-A and VE-cadherin, using vinculin as loading control.Quantification of data presented in A. JAM-A expression levels were normalized with vinculin and are expressed as percentages. Data are means ± s.d. from three independent experiments.HUVECs with stable scrambled shRNA (ctrl) or a JAM-A targeting shRNA (#51) were seeded onto Transwell filters for 72 h. 6-CFDA-labeled human MABs derived from three different donors (22-, 42-and 37-year old [y.o.] healthy donors) were added to the upper chamber and allowed to migrate for 8 h. Migrated MABs on the lower sides of the filters (green) were fixed and counted. Representative data are shown from four independent experiments, each in triplicate. Scale bar: 100 μm.Quantification of migrated MABs per area is shown for 22 y.o. (left), 42 y.o. (middle) and 37 y.o. (right) MABs. * P < 0.01, ** P < 0.001. Data are means ± s.e.m. from four independent experiments, each in triplicate.Confluent monolayers of HUVECs were treated with non-related IgG, J10.4 and BV16 (12 μg/ml) at 37°C, for the indicated times. The cells were then incubated for the last 1 h with 100 μg/ml cycloheximide (chx) to inhibit protein synthesis, fixed and stained for JAM-A (red) and VE-cadherin (marker for junctions, green). Scale bar: 20 μm.Quantification of JAM-A delocalization. Time course (1, 3, 6 and 15 h) for mean fluorescence (expressed in arbitrary units, AU, in cells treated with IgG, J10.4 and BV16, as indicated). The trend lines are shown. *** P < 0.00001 compared with IgG. Data are means ± s.e.m. from three independent experiments.Experiments performed as in C, except the HUVECs were pre-treated (3 h) and further incubated (8 h) with non-related IgG, J10.4 and BV16 (as indicated). Quantification of migrated MABs per area is shown for 22 y.o. (left), 42 y.o. (middle) and 37 y.o. (right) MABs. ** P < 0.001. Data are means ± s.e.m. from three independent experiments, each in triplicate.Source data are available for this figure.
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fig04: HUVECs with stable scrambled shRNA (Ctrl) or JAM-A targeting shRNAs (#51, #49, #50, #52) were generated (see Materials and Methods) and then homogenized. The cell lysates were analyzed by immunoblotting for JAM-A and VE-cadherin, using vinculin as loading control.Quantification of data presented in A. JAM-A expression levels were normalized with vinculin and are expressed as percentages. Data are means ± s.d. from three independent experiments.HUVECs with stable scrambled shRNA (ctrl) or a JAM-A targeting shRNA (#51) were seeded onto Transwell filters for 72 h. 6-CFDA-labeled human MABs derived from three different donors (22-, 42-and 37-year old [y.o.] healthy donors) were added to the upper chamber and allowed to migrate for 8 h. Migrated MABs on the lower sides of the filters (green) were fixed and counted. Representative data are shown from four independent experiments, each in triplicate. Scale bar: 100 μm.Quantification of migrated MABs per area is shown for 22 y.o. (left), 42 y.o. (middle) and 37 y.o. (right) MABs. * P < 0.01, ** P < 0.001. Data are means ± s.e.m. from four independent experiments, each in triplicate.Confluent monolayers of HUVECs were treated with non-related IgG, J10.4 and BV16 (12 μg/ml) at 37°C, for the indicated times. The cells were then incubated for the last 1 h with 100 μg/ml cycloheximide (chx) to inhibit protein synthesis, fixed and stained for JAM-A (red) and VE-cadherin (marker for junctions, green). Scale bar: 20 μm.Quantification of JAM-A delocalization. Time course (1, 3, 6 and 15 h) for mean fluorescence (expressed in arbitrary units, AU, in cells treated with IgG, J10.4 and BV16, as indicated). The trend lines are shown. *** P < 0.00001 compared with IgG. Data are means ± s.e.m. from three independent experiments.Experiments performed as in C, except the HUVECs were pre-treated (3 h) and further incubated (8 h) with non-related IgG, J10.4 and BV16 (as indicated). Quantification of migrated MABs per area is shown for 22 y.o. (left), 42 y.o. (middle) and 37 y.o. (right) MABs. ** P < 0.001. Data are means ± s.e.m. from three independent experiments, each in triplicate.Source data are available for this figure.

Mentions: We then extended the study to human cells. For this, we first generated human umbilical vein endothelial cells (HUVECs) that were stably infected with lentiviral vectors expressing shRNA against JAM-A. Four different shRNA constructs were tested to establish stable JAM-A deficient cell lines. The efficiency of the different constructs was evaluated using Western blot (Fig 4A) and the relative densitometry showed that sh#50 and sh#51 RNAs significantly reduced JAM-A protein expression by approximately 75–85%, as compared to the control (Fig 4B). The sh#50, sh#51 and sh#52 RNAs were then selected to assess the impact of JAM-A down-regulation on human MAB transmigration. The human MABs were derived from three healthy donors and were selected for their different spontaneous myogenic differentiation into skeletal myosin heavy chain positive-myotubes (supplementary Fig S2C). Moreover, as we previously reported for murine MABs, Western blot analysis showed only a faint band corresponding to JAM-A in 37 years old (y.o.) human MABs, while 22 y.o. and 42 y.o. MABs did not express JAM-A (supplementary Fig S2B, right panel). Consistent with the data obtained with murine cells, the human MABs migrated more efficiently when the endothelial JAM-A was reduced and the increase in cell transmigration correlated with the efficiency of JAM-A depletion in HUVECs, suggesting a dose-dependent effect (Fig 4B-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)

HUVECs with stable scrambled shRNA (Ctrl) or JAM-A targeting shRNAs (#51, #49, #50, #52) were generated (see Materials and Methods) and then homogenized. The cell lysates were analyzed by immunoblotting for JAM-A and VE-cadherin, using vinculin as loading control.Quantification of data presented in A. JAM-A expression levels were normalized with vinculin and are expressed as percentages. Data are means ± s.d. from three independent experiments.HUVECs with stable scrambled shRNA (ctrl) or a JAM-A targeting shRNA (#51) were seeded onto Transwell filters for 72 h. 6-CFDA-labeled human MABs derived from three different donors (22-, 42-and 37-year old [y.o.] healthy donors) were added to the upper chamber and allowed to migrate for 8 h. Migrated MABs on the lower sides of the filters (green) were fixed and counted. Representative data are shown from four independent experiments, each in triplicate. Scale bar: 100 μm.Quantification of migrated MABs per area is shown for 22 y.o. (left), 42 y.o. (middle) and 37 y.o. (right) MABs. * P < 0.01, ** P < 0.001. Data are means ± s.e.m. from four independent experiments, each in triplicate.Confluent monolayers of HUVECs were treated with non-related IgG, J10.4 and BV16 (12 μg/ml) at 37°C, for the indicated times. The cells were then incubated for the last 1 h with 100 μg/ml cycloheximide (chx) to inhibit protein synthesis, fixed and stained for JAM-A (red) and VE-cadherin (marker for junctions, green). Scale bar: 20 μm.Quantification of JAM-A delocalization. Time course (1, 3, 6 and 15 h) for mean fluorescence (expressed in arbitrary units, AU, in cells treated with IgG, J10.4 and BV16, as indicated). The trend lines are shown. *** P < 0.00001 compared with IgG. Data are means ± s.e.m. from three independent experiments.Experiments performed as in C, except the HUVECs were pre-treated (3 h) and further incubated (8 h) with non-related IgG, J10.4 and BV16 (as indicated). Quantification of migrated MABs per area is shown for 22 y.o. (left), 42 y.o. (middle) and 37 y.o. (right) MABs. ** P < 0.001. Data are means ± s.e.m. from three independent experiments, each in triplicate.Source data are available for this figure.
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fig04: HUVECs with stable scrambled shRNA (Ctrl) or JAM-A targeting shRNAs (#51, #49, #50, #52) were generated (see Materials and Methods) and then homogenized. The cell lysates were analyzed by immunoblotting for JAM-A and VE-cadherin, using vinculin as loading control.Quantification of data presented in A. JAM-A expression levels were normalized with vinculin and are expressed as percentages. Data are means ± s.d. from three independent experiments.HUVECs with stable scrambled shRNA (ctrl) or a JAM-A targeting shRNA (#51) were seeded onto Transwell filters for 72 h. 6-CFDA-labeled human MABs derived from three different donors (22-, 42-and 37-year old [y.o.] healthy donors) were added to the upper chamber and allowed to migrate for 8 h. Migrated MABs on the lower sides of the filters (green) were fixed and counted. Representative data are shown from four independent experiments, each in triplicate. Scale bar: 100 μm.Quantification of migrated MABs per area is shown for 22 y.o. (left), 42 y.o. (middle) and 37 y.o. (right) MABs. * P < 0.01, ** P < 0.001. Data are means ± s.e.m. from four independent experiments, each in triplicate.Confluent monolayers of HUVECs were treated with non-related IgG, J10.4 and BV16 (12 μg/ml) at 37°C, for the indicated times. The cells were then incubated for the last 1 h with 100 μg/ml cycloheximide (chx) to inhibit protein synthesis, fixed and stained for JAM-A (red) and VE-cadherin (marker for junctions, green). Scale bar: 20 μm.Quantification of JAM-A delocalization. Time course (1, 3, 6 and 15 h) for mean fluorescence (expressed in arbitrary units, AU, in cells treated with IgG, J10.4 and BV16, as indicated). The trend lines are shown. *** P < 0.00001 compared with IgG. Data are means ± s.e.m. from three independent experiments.Experiments performed as in C, except the HUVECs were pre-treated (3 h) and further incubated (8 h) with non-related IgG, J10.4 and BV16 (as indicated). Quantification of migrated MABs per area is shown for 22 y.o. (left), 42 y.o. (middle) and 37 y.o. (right) MABs. ** P < 0.001. Data are means ± s.e.m. from three independent experiments, each in triplicate.Source data are available for this figure.
Mentions: We then extended the study to human cells. For this, we first generated human umbilical vein endothelial cells (HUVECs) that were stably infected with lentiviral vectors expressing shRNA against JAM-A. Four different shRNA constructs were tested to establish stable JAM-A deficient cell lines. The efficiency of the different constructs was evaluated using Western blot (Fig 4A) and the relative densitometry showed that sh#50 and sh#51 RNAs significantly reduced JAM-A protein expression by approximately 75–85%, as compared to the control (Fig 4B). The sh#50, sh#51 and sh#52 RNAs were then selected to assess the impact of JAM-A down-regulation on human MAB transmigration. The human MABs were derived from three healthy donors and were selected for their different spontaneous myogenic differentiation into skeletal myosin heavy chain positive-myotubes (supplementary Fig S2C). Moreover, as we previously reported for murine MABs, Western blot analysis showed only a faint band corresponding to JAM-A in 37 years old (y.o.) human MABs, while 22 y.o. and 42 y.o. MABs did not express JAM-A (supplementary Fig S2B, right panel). Consistent with the data obtained with murine cells, the human MABs migrated more efficiently when the endothelial JAM-A was reduced and the increase in cell transmigration correlated with the efficiency of JAM-A depletion in HUVECs, suggesting a dose-dependent effect (Fig 4B-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