Flt-1 haploinsufficiency ameliorates muscular dystrophy phenotype by developmentally increased vasculature in mdx mice

Abstract

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disease caused by mutations in the gene coding for the protein dystrophin. Recent work demonstrates that dystrophin is also found in the vasculature and its absence results in vascular deficiency and abnormal blood flow. This induces a state of ischemia further aggravating the muscular dystrophy pathogenesis. For an effective form of therapy of DMD, both the muscle and the vasculature need to be addressed. To reveal the developmental relationship between muscular dystrophy and vasculature, mdx mice, an animal model for DMD, were crossed with Flt-1 gene knockout mice to create a model with increased vasculature. Flt-1 is a decoy receptor for vascular endothelial growth factor, and therefore both homozygous (Flt-1(-/-)) and heterozygous (Flt-1(+/-)) Flt-1 gene knockout mice display increased endothelial cell proliferation and vascular density during embryogenesis. Here, we show that Flt-1(+/-) and mdx:Flt-1(+/-) adult mice also display a developmentally increased vascular density in skeletal muscle compared with the wild-type and mdx mice, respectively. The mdx:Flt-1(+/-) mice show improved muscle histology compared with the mdx mice with decreased fibrosis, calcification and membrane permeability. Functionally, the mdx:Flt-1(+/-) mice have an increase in muscle blood flow and force production, compared with the mdx mice. Consequently, the mdx:utrophin(-/-):Flt-1(+/-) mice display improved muscle histology and significantly higher survival rates compared with the mdx:utrophin(-/-) mice, which show more severe muscle phenotypes than the mdx mice. These data suggest that increasing the vasculature in DMD may ameliorate the histological and functional phenotypes associated with this disease.

Combined Therapy with Sonic Hedgehog Gene Transfer and Bone Marrow-Derived Endothelial Progenitor Cells Enhances Angiogenesis and Myogenesis in the Ischemic Skeletal Muscle

“…We have already demonstrated that Shh is able to stimulate the process of myogenesis after injury of the skeletal muscle in vivo [4], and other studies in the literature report that Shh increases myoblast proliferation in vitro [18,19,20,21]. The fact that such myogenic effects are enhanced by phShh + EPC combination therapy is consistent with the concept that angiogenesis and myogenesis are interdependent mechanisms and that the vascular niche promotes the proliferation of myoblasts [16,39]. The increased number of proliferating myoblasts and regenerating fibers observed upon combined treatment with phShh and EPCs might be the result of increased myoblast proliferation, promoted directly by the endothelial cells or indirectly by factors produced by them [16,39].…”

“…It has been demonstrated that endothelial cells support the proliferation of myoblasts in vitro [16]. Here, we cultured satellite cell-derived primary myoblasts in the presence or absence of BM-derived EPCs and found increased myoblast proliferation in cultures also containing EPCs.…”

“…Myoblasts were cultured in myoblast growth medium, as described [16], with or without EPCs and with or without the addition of 10 µg/ml of Shh recombinant protein (R&D Systems). Myoblast proliferation was quantified by the number of desmin-positive cells 3 days after coculture, as described [16]. At least 100 desmin-positive myoblasts were counted for each experiment.…”

Structural and Functional Alterations of Skeletal Muscle Microvasculature in Dystrophin-Deficient mdx Mice

“…A previous study demonstrated that mdx mice knockout for the Flt1 receptor (mdx:Flt1 þ/À ) presented an improved muscle histology associated with a better muscle perfusion and force production compared with mdx mice. 34 These data underline the link between vascular remodeling and muscle regeneration, even in severe chronic diseases. Concerning VEGF, the twofold decreased expression, detected in old mdx mice, was in accordance with previous studies demonstrating that treatment with VEGF strongly ameliorates the mdx phenotype, with improvement of functional parameters, an increase in capillary density, improved muscle regeneration, and a decrease in interstitial fibrosis.…”

Nitric oxide donors improve prednisone effects on muscular dystrophy in themdxmouse diaphragm

“…Continuous mechanical activity by the diaphragm (5, 54) further modulates disease severity, as fibers are prone to early contraction-induced damage in the absence of dystrophin (68,79). However, since respiratory insufficiency occurs as a late sequel and is one of the most common causes of death in dystrophy (33), the systemic effects of NO donor formulations in muscular dystrophy, including sildenafil (47), require further study, particularly in relation to the potential to improve vascular perfusion during exercise (11,72,86) and the potential to reduce disease progression and offset sequelae of steroid treatment. Similar effects of NO donors on muscle perfusion in old animals merit investigation for potential application to treating age-related atrophy.…”