We have shown that the Sonic Hedgehog (Shh) embryonic signalling pathway can be reactivated in myocardial infarction (MI) in adults inducing expression of pro-angiogenic factors. We hypothesized that combining Shh gene therapy and endothelial BM-derived pro-angiogenic cell mobilization by a CXCR4 antagonist, AMD3100 (AMD), could exert synergistic effects and would be superior to either single strategy for the treatment of MI.
Methods and Results
In vitro, human Shh plasmid activates the Hedgehog pathway and induces transcription and secretion of VEGF and SDF-1 from fibroblasts. Shh conditioned medium from these fibroblasts promotes capillary morphogenesis, proliferation and migration. In vivo, MI was induced in WT and GFP-bone marrow (BM) transplanted mice randomly assigned in 4 treatment groups: control ; AMD (single dose, 5mg/kg s.c.); Shh (intramyocardial administration of 100μg Shh plasmid DNA at time of MI surgery); AMD+Shh group. Left ventricular ejection fraction (LVEF) was evaluated by echo up to 4 weeks post MI. AMD+Shh group exhibited the best LV function (control: 28+/-3 % ; AMD: 36+/-2 % ; Shh: 40+/-3 % ; AMD+Shh: 48+/-2 % ; P<0.05 AMD+Shh vs. other groups). Furthermore, combination of AMD with sub-therapeutic dose of Shh resulted in a significant improvement of cardiac function recovery compared to monotherapy, highlighting its synergistic effect (P<0.05). Elastic staining and immunohistological analyses demonstrated reduced infarct size and increased capillary density in the AMD+Shh group (both P<0.05). Combination therapy was also associated with significant increase in number of GFP-BS lectin BM-derived cells incorporated into the ischemic area (P<0.05).
We then explored the certain potential mechanisms of the favourable effects of combination therapy. MMP-9 mRNA expression was increased in ischemic myocardium in the AMD+Shh (10-fold versus control). The positive effect on EF of combined treatment was attenuated in MMP-9 KO mice (WT mice 49+/-2 % vs. KO mice 36+/-4 % ; P<0.05), suggesting that MMP-9 might be a key modulator of the combination therapy.
Pharmacological enhancement of Shh gene therapy via BM-cell mobilization by a CXCR4 antagonist is mediated via an MMP-9-dependent pathway. The combination may offer advantages in safety and feasibility by allowing lower dose gene transfer while improving outcomepost-MILe texte complet de cet article est disponible en PDF.