Targeting endothelial junctional adhesion molecule-A/ EPAC/ Rap-1 axis as a novel strategy to increase stem cell engraftment in dystrophic muscles.
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.
Affiliation: FIRC Institute of Molecular Oncology Foundation (IFOM), Milan, Italy.
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.
- Cell Adhesion Molecules/antagonists & inhibitors/deficiency/metabolism*
- Guanine Nucleotide Exchange Factors/metabolism*
- Muscular Dystrophies/pathology/surgery/therapy*
- Receptors, Cell Surface/antagonists & inhibitors/deficiency/metabolism*
- Signal Transduction*
- Stem Cell Transplantation*
- Stem Cells/cytology*
- rap1 GTP-Binding Proteins/metabolism*
- Cell Movement
- Endothelial Cells/metabolism/pathology
- Endothelium, Vascular/metabolism/pathology
- Muscle, Skeletal/injuries/pathology
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fig07: JAM-A-WT endothelial cells were starved for 16 h and then treated with vehicle (ctrl), a selective activator of EPAC (007, 100 μM) or tetradecanoylphorbol acetate (TPA, 10 μM), an activator of the signal transduction enzyme protein kinase C, used as positive control for Rap-1 activation. Rap1-GTP loading was quantified by GST pull-down/ Western blotting and densitometry analysis. Representative blot (left) and quantification of densitometry analysis (right) of Rap-1-GTP levels normalized for total amount of protein. ** P < 0.001. Data are means ± s.d. from three independent experiments.JAM-A-WT endothelial cells were incubated with the Rap-1 pharmacological inhibitor GGTI-298 (10 μM) or vehicle for 3 h and 6 h (as indicated). Rap1-GTP loading was quantified as in A. Data are means ± s.d. from three independent experiments. Note: to highlight Rap-1 activation, the filter in A was exposed for 30 s, while in B it was exposed for 5 min, to improve the evaluation of Rap-1 inhibition at 3 h and 6 h.JAM-A-WT and JAM-A- endothelial cells seeded on coated filters for 72 h and treated with vehicle (ctrl) or 007 or GGTI-298 for the last 30 min, as indicated. 6-CFDA-labelled adult MABs were added to the upper chamber and allowed to migrate for 6 h under these conditions. Quantification of migrated MABs per area for JAM-A-WT (on the left of the dashed line) and JAM-A- (on the right of the dashed line) endothelial cells. ** P < 0.001. Data are means ± s.e.m. from three independent experiments, each in triplicate.6-CFDA-labelled embryonic (left) and adult (right) MABs were incubated with vehicle (ctrl, white bars), 007 (100 μM) or GGTI-298 (10 μM) (black bars) for 3 h, as indicated. 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.HUVECs were treated as described in C. 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.Sgca- mice were treated with GGTI-298 (50 mg/Kg, n = 2) or with vehicle (ctrl, n = 3) for 1 h and then were intra-arterially transplanted with adult MABs. After 6 h, the hind limb muscles were collected and the presence of migrated cells was quantified using qRT-PCR with nLacZ primers. The relative RNA level of nLacZ obtained for control was set to 1, and the ratio for GGTI-298 versus control is shown. Fold increases have been extrapolated by data shown in Figure S1H.Box-plot showing exercise tolerance in a treadmill test for untreated Sgca-/scid/beige mice, Sgca-/scid/beige mice transplanted with MABs (+ MABs, 5 × 105 cells injected bilaterally in femoral arteries) and Sgca-/scid/beige mice transplanted with MABs and treated with GGTI-298 (+MABs + GGTI-298, 5 × 105 cells injected bilaterally in femoral arteries). Values are plotted as fold increase of motor capacity measured at different time points (21, 28 and 35 days post transplantation), and were normalized with the baseline performances of each mouse. Data are means ± s.e.m. of 3 mice per group. ** P < 0.01, *** P < 0.001, one-way ANOVA.Source data are available for this figure.
As the data above show that JAM-A acts by stimulating Rap-1 activity, we asked whether also the trans-endothelial migration of MABs could be modulated by Rap-1. To address this question, we used the selective activator of EPAC, 007, and the Rap-1 pharmacological inhibitor GGTI-298. As shown in Fig 7A, Rap-1-GTP increased in the presence of 007, as also seen for tetradecanoylphorbol acetate (TPA), an activator of the signal transduction enzyme protein kinase C used here as a positive control. On the other hand, Rap-1 activation was strongly inhibited in the presence of GGTI-298 (Fig 7B). Following Rap-1 activation via 007, the transmigration of adult murine MABs was significantly reduced by some 60% (Fig 7C). Conversely, the transmigration of MABs was significantly doubled in the presence of the Rap-1 inhibitor GGTI-298, which suggested an active contribution of Rap-1 in MAB extravasation. Interestingly, incubation of the JAM-A- endothelial cells with 007 significantly counteracted the ability of MABs to migrate through the endothelium (50% inhibition), which supports the concept that Rap-1 might by-pass JAM-A in inhibiting MAB transmigration (Fig 7C). Conversely, GGTI-298 treatment had no effect on JAM-A- endothelial cells (Fig 7C).