Vascular endothelial growth factor reduced hypoxia-induced death of human myoblasts and improved their engraftment in mouse muscles.

Muscle precursor cell (myoblasts) transplantation is considered as a potential approach to restore dystrophin expression in Duchenne muscular dystrophy (DMD) patients. The study purpose was to verify the implication of hypoxia in the myoblast death observed after their transplantation and also to evaluate the potential beneficial effects of vascular endothelial growth factor (VEGF) overexpression on myoblast engraftment in a murine model. Pimonidazole hydrochloride (hypoxyprobe-1) was used to mark selectively myoblasts to evaluate their hypoxia in vivo. In vitro, hypoxia was induced by culturing human myoblasts in hypoxic environment. In vitro effects of VEGF(165) on survival of human cells was assessed by Hoescht-PI labeling. Tibialis anterior (TA) female mouse muscles were electroporated with a plasmid containing the VEGF(165) or with an empty vector. Circulating VEGF concentration was assessed by ELISA. After 2 weeks of electroporation, severe combined immunodeficient (SCID) mice were transplanted with 800 000 human male myoblasts labeled with radioactive thymidine. Mouse muscles were harvested 2 and 4 days later and myoblast survival and proliferation were evaluated by scintigraphy and Y chromosome quantitative PCR. The long-term graft success was evaluated using gamma-radiograph imaging and by counting the dystrophin positive muscle fibers. Hypoxyprobe labeling has shown that most of the transplanted myoblasts were hypoxic. The transplantation of radioactive male myoblasts in female mice electroporated with the VEGF(165) plasmid demonstrated that VEGF reduced their death by 10% but did not improve their proliferation. VEGF(165) enhanced human myoblast survival in vitro under hypoxic conditions. Electroporation of TA muscles of SCID mouse with the vector coding for VEGF(165) promoted angiogenesis and improved by 1.5-fold the success of myoblast transplantation in comparison with the control mice that were electroporated with the empty vector. These results indicate that hypoxia is partially responsible for the death of the transplanted myoblasts. VEGF can be used to improve myoblast survival and the graft success.

Overexpression of follistatin in human myoblasts increases their proliferation and differentiation, and improves the graft success in SCID mice.

Duchenne muscular dystrophy is caused by the absence of functional dystrophin, leading to the myofiber membrane instability and progressive muscle atrophy. Myoblast transplantation in dystrophic muscles is a potential therapy, as it permits the long-term restoration of dystrophin expression in transplanted muscles. However, the success of this approach is limited by the short period of muscle repair following myoblast transplantation. Myostatin, a powerful inhibitor of muscle growth, is involved in terminating the period of muscle repair following injury by reducing myoblast proliferation and differentiation. Follistatin forms a complex with myostatin, preventing its interaction with its receptor and thus blocking the myostatin signal. Here, we used a lentivirus to overexpress the follistatin protein in normal myoblasts to block the myostatin signaling. We measured the potential of transduced myoblasts to proliferate and to form multinucleated myotubes in vitro. And finally, we considered the engraftment success of those transduced myoblasts in comparison with control cells in vivo within SCID mice TA muscle. Our results first confirmed the overexpression of follistatin into lentivirus transduced myoblasts, and second showed that the overexpression of the follistatin in normal human myoblasts improved in vitro their proliferation rate by about 1.5-fold after 96 h and also their differentiation rate by about 1.6- and 1.8-fold, respectively, in the absence and in the presence of recombinant myostatin. Finally, our data demonstrated that the engraftment of human normal myoblasts overexpressing the follistatin protein into SCID mouse muscles was enhanced by twofold.

Induction of Anoikis following myoblast transplantation into SCID mouse muscles requires the Bit1 and FADD pathways.

Seventy-five percent of the myoblasts transplanted in the mouse muscle die during the first 4 days following transplantation. The purpose of this study was to determine if anoikis plays a role in this phenomenon. Survival and proliferation of myoblasts in vitro were determined by Hoescht-PI labeling and cell counts respectively. In vivo cell survival and proliferation were quantified by injecting human male myoblasts labeled with (14)C-thymidine in SCID mouse muscles. Survival and proliferation of the transplanted myoblasts were evaluated by scintigraphy and quantitative PCR of human Y chromosomal DNA. Inclusion of the extracellular matrix protein fibronectin enhanced transplanted myoblast survival by 1.7-fold while vitronectin improved their proliferation by 1.8-fold. Reductions in FADD and Bit1 expression reduced anoikis in vitro and improved the injected myoblast survival in vivo. Ectopic expression of the anti-apoptotic protein Bcl-2 completely abolished myoblast anoikis in vitro and enhanced cell survival by 3.1-fold in vivo. Cell death following transplantation appears to me mediated in part by anoikis. Inclusion of extracellular matrix proteins enhanced both survival and proliferation. Reduced expression of the proapoptotic proteins Bit1 and FADD or overexpression of Bcl-2 improved myoblast survival.

Tubulyzine, a novel tri-substituted triazine, prevents the early cell death of transplanted myogenic cells and improves transplantation success.

Myoblast transplantation (MT) is a potential therapeutic approach for several muscular dystrophies. A major limiting factor is that only a low percentage of the transplanted myoblasts survives the procedure. Recent advances regarding how and when the myoblasts die indicate that events preceding actual tissue implantation and during the first days after the transplantation are crucial. Myoseverin, a recently identified tri-substituted purine, was shown to induce in vitro the fission of multinucleated myotubes and affect the expression of a variety of growth factors, and immunomodulation, extracellular matrix-remodeling, and stress response genes. Since the effects of myoseverin are consistent with the activation of pathways involved in wound healing and tissue regeneration, we have investigated whether pretreatment and co-injection of myoblasts with Tubulyzine (microtubule lysing triazine), an optimized myoseverin-like molecule recently identified from a triazine library, could reduce myoblast cell death following their transplantation and consequently improves the success of myoblast transplantation. In vitro, using annexin-V labeling, we showed that Tubulyzine (5 microM) prevents normal myoblasts from apoptosis induced by staurosporine (1 microM). In vivo, the pretreatment and co-injection of immortal and normal myoblasts with Tubulyzine reduced significantly cell death (assessed by the radio-labeled thymidine of donor DNA) and increased survival of myoblasts transplanted in Tibialis anterior (TA) muscles of mdx mice, thus giving rise to more hybrid myofibers compared to transplanted untreated cells. Our results suggest that Tubulyzine can be used as an in vivo survival factor to improve the myoblast-mediated gene transfer approach.