Age-dependent dysregulation of muscle vasculature and blood flow recovery after hindlimb ischemia in the mdx model of duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD), caused by a lack of functional dystrophin, is characterized by progressive muscle degeneration. Interestingly, dystrophin is also expressed in endothelial cells (ECs), and insufficient angiogenesis has already been hypothesized to contribute to DMD pathology, however, its status in mdx mice, a model of DMD, is still not fully clear. Our study aimed to reveal angiogenesis-related alterations in skeletal muscles of mdx mice compared to wild-type (WT) counterparts. By investigating 6-and 12-week-old mice, we sought to verify if those changes are age-dependent. We utilized a broad spectrum of methods ranging from gene expression analysis, flow cytometry, and immunofluorescence imaging to determine the level of angiogenic markers and to assess muscle blood vessel abundance. Finally, we implemented the hindlimb ischemia (HLI) model, more biologically relevant in the context of functional studies evaluating angiogenesis/arteriogenesis processes. We demonstrated that both 6-and 12-week-old dystrophic mice exhibited dysregulation of several angiogenic factors, including decreased vascular endothelial growth factor A (VEGF) in different muscle types. Nonetheless, in younger, 6-week-old mdx animals, neither the abundance of CD31+α-SMA+ double-positive blood vessels nor basal blood flow and its restoration after HLI was affected. In 12-week-old mdx mice, although a higher number of CD31+αSMA+ double-positive blood vessels and an increased percentage of skeletal muscle ECs were found, the abundance of pericytes was diminished, and blood flow was reduced. Moreover, impeded perfusion recovery after HLI associated with a blunted inflammatory and regenerative response was evident in 12-week-old dystrophic mice. Hence, our results reinforce the hypothesis of age-dependent angiogenic dysfunction in dystrophic mice. In conclusion, we suggest that older mdx mice constitute an appropriate model for preclinical studies evaluating the effectiveness of vascular-based therapies aimed at the restoration of functional angiogenesis to mitigate DMD severity.