Targeting vascular endothelium for muscular dystrophy therapy

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ABSTRACT Skeletal muscle is a highly ordered, yet complex tissue which contains several cell types that interact with each other to maintain structure and homeostasis. Muscle satellite cells (SCs) are a stem cell population responsible for muscle growth, repair and regeneration. Maintenance of the balance between SC differentiation and self- renewal is required for muscle homeostasis. A defect in self-renewal ability leads to a decrease in SC number, resulting in depletion of the SC pool and reduced muscle regeneration capacity, which occurs in aged and diseased muscle, including Duchenne muscular dystrophy (DMD). DMD is a progressive disorder in which the absence of the dystrophin protein results in loss of the dystrophin bridge at the muscle membrane. Recent work demonstrates the angiogenic impairment of the ECs in DMD model mdx mice. We demonstrates the increased vascular density can ameliorate phenotypes associated with DMD. However, the importance of angiogenesis in DMD treatment has not yet been well addressed. A recent study demonstrated that SCs are preferentially located next to capillary endothelial cells (ECs). However, the exact relationship between SCs and ECs has yet to be examined. Recently, we established an optimized tissue clearing protocol for skeletal muscle. We utilized fluorescent reporters for SCs and ECs along with tissue clearing to demonstrate the close proximity of SCs to capillaries in 3-dimentional imaging, suggesting the juxtavascular niche of SCs for stem cell maintenance. SCs express vascular endothelial cell growth factor (VEGF) and Notch receptors which is dynamically altered during the regeneration process. Therefore, we hypothesize that SCs recruit capillary ECs via VEGF to establish a juxtavascular niche for SC maintenance during homeostasis and regeneration. In this proposal, we will determine the extent to which an increased vascular niche in postnatal conditional knockout of Flt1 and by blocking Flt1 via anti-Flt1 nanobodies, created by a combination of llama-immunization and phage display technologies, in mdx mice can increase SC number, reduced pathological endothelial-to-mesenchymal transition (EndMT), and ameliorate phenotypes associated with DMD. Consequently, we will elucidate key molecules that regulate angio- myogenesis, including those involved in SC homeostasis, which is fundamental to developing therapeutic approaches to treat DMD. SCs and their juxtavascular niche are potential therapeutic targets to induce and maintain SC populations that slow the loss of skeletal muscle function with DMD.