Age-Dependent Dysregulation of Muscle Vasculature and Blood Flow Recovery after Hindlimb Ischemia in the mdx Model of Duchenne Muscular Dystrophy.

Paulina Podkalicka,Józef Dulak,Kalina Andrysiak,Mateusz Tomczyk,Agnieszka Łoboda,Katarzyna Kaziród,Olga Mucha,Iwona Bronisz-Budzyńska,Jacek Stępniewski,Sophie Ostrowska-Paton

doi:10.3390/biomedicines9050481

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+α-SMA+ 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.

Highlights

The mdx phenotype is considered mild compared to humans [34], several features of the disease are well recapitulated in those animals, as reported in our recent studies [22,23,24,25]
Dystrophic mice exhibited decreased muscle functionality in a forelimb grip strength test (Figure S1a), which was accompanied by increased body weight (Figure S1b) and an abundance of CD45− CD31− Sca-1+ CD34+ fibroadipogenic progenitors (FAPs) evaluated by flow cytometry in the gastrocnemius muscle (Figures S1c and S2)
Diminished expression of vascular endothelial growth factor A (VEGF) was observed in dystrophic skeletal muscle cells differentiated from human induced pluripotent stem cells (Figure 1g)

Summary

Introduction

Duchenne muscular dystrophy (DMD), a genetic disease that predominantly affects boys already in early childhood, represents one of the most destructive types of muscular dystrophies. It is caused by a lack of functional dystrophin, as the result of more than 7000 patient-specific mutations in one of the largest human genes, DMD, containing 79 exons and approximately 2.4 million bp [1,2]. Dystrophin with its protein partners forms a large dystrophin–glycoprotein complex, abundantly expressed in the sarcolemma. Its absence is associated with higher susceptibil-

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