Modulating JAM-A Expression to Enhance Mesoangioblast Diapedesis: An In Vitro Investigation

Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disorder caused by mutations in the dystrophin gene. The absence of functional dystrophin leads to muscle fiber instability, progressive degeneration, and fibro-adipose replacement. Despite multidisciplinary care, current treatments remain largely palliative, and DMD represents a significant unmet medical need. Among the investigated therapeutic strategies, cell therapy using mesoangioblasts (MABs) has shown promise. MABs are vessel-associated multipotent progenitors capable of crossing the endothelial barrier and homing to dystrophic muscles, where they contribute to regeneration by fusing with myofibers. A "first-in-human" Phase I/II clinical trial using HLA-matched donor hMABs demonstrated safety but limited clinical efficacy, primarily due to low cell engraftment. Subsequent research identified Junctional Adhesion Molecule-A (JAM-A), a key component of endothelial tight junctions, as a critical regulator of MAB diapedesis. Previous preclinical studies demonstrated that downregulation of JAM-A with neutralizing antibody BV11 could significantly enhance MAB transmigration. Subsequently, a combined approach using MABs transduced with a lentivector expressing the U7 snRNA for exon 51 skipping was developed. The U7 snRNA spreads along the regenerating myofiber and enters the host’s neighboring nuclei, potentially achieving therapeutic levels of dystrophin even with modest engraftment. The aim of this study was to evaluate clinically approved drugs for their ability to induce a transient downregulation of JAM-A, providing a translatable drugs-repositioning strategy. Three candidates—rosiglitazone, pioglitazone, and metformin—were screened using a 2D endothelial model (HUVECs). Initial immunofluorescence screening at various concentrations and time points identified metformin as the most suitable compound due to its efficacy and favorable safety profile. The reduction of JAM-A expression was further validated through Western Blot and RT-PCR analyses. Finally, the functional impact of metformin-mediated JAM-A modulation was assessed using a Boyden chamber transmigration assay. U7#51T2AGFP hMABs were challenged to migrate through HUVEC monolayers toward an SDF-1 gradient and paracrine stimuli from C2C12 myotubes. The results showed a significant increase in MAB diapedesis in metformin-treated conditions, with an approximately twofold increase in transmigrated cells compared to controls. These findings suggest that metformin-mediated modulation of JAM-A represents a feasible and clinically translatable strategy to enhance the extravasation and therapeutic potential of MABs in DMD cell-based therapies. Further in vivo studies will be essential to confirm whether this combined approach improves long-term cell engraftment and dystrophin restoration.