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

Abstract

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 g-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.

Hypoxic preconditioning enhances bone marrow mesenchymal stem cell migration via Kv2.1 channel and FAK activation

“…For example, we and others have shown that overexpression of the anti-apoptotic gene bcl-2 or bcl-X L enhances cell viability after transplantation into the ischemic brain or damaged spinal cord (24,52). Trophic factors such as vascular endothelial growth factor (VEGF) coapplied with transplanted cells may have protective effects and augment therapeutic benefits (3,38). In our recent investigations, we tested a new strategy of preconditioning cells before transplantation with sublethal hypoxia.…”

Mechanical Loading of Stem Cells for Improvement of Transplantation Outcome in a Model of Acute Myocardial Infarction: The Role of Loading History

“…18 Previous reports suggest that VEGF influences myoblast migration and survival and increases skeletal muscle repair during ischemia. 42,43 Kasper et al showed that the conditioned medium from mesenchymal stem cells subjected to mechanical loading increased sprouting of endothelial cells by creating an angiogenic promoting environment. 44 Certainly other paracrine factors besides VEGF could also be playing a role in the improvement of cardiac function imparted by the MS MDSCs.…”

Testosterone protects from metabolic syndrome-associated prostate inflammation: an experimental study in rabbit

“…Pimonidazole (hypoxyprobe) is a water soluble substituted 2-nitrominidazole that is rapidly distributed to all tissues in the body, but it only forms adducts with proteins in hypoxic cells, which are at an oxygen pressure equal to or lower than 10 mmHg. Hypoxic cells are thereafter easily detected by immunohistochemistry, using a monoclonal antibody (Hypoxyprobe-1Mab1) that recognizes hypoxyprobe protein adduct (Durand & Raleigh 1998, Bennewith et al 2002; Pimonidazole as a probe for the in vivo detection of hypoxia has been validated previously (Arteel et al 1995, Vignozzi et al 2006, 2008, 2009, Bouchentouf et al 2008, Via et al 2008, Morelli et al 2010a. Rabbit ventral prostate samples were rapidly removed and fixed in 4% neutral buffered formalin, dehydrated, and embedded in paraffin.…”