Research Article: ANG1 treatment reduces muscle pathology and prevents a decline in perfusion in DMD mice

Date Published: March 23, 2017

Publisher: Public Library of Science

Author(s): Kelly M. Gutpell, Nikola Tasevski, Boaz Wong, William Thomas Hrinivich, Feng Su, Jennifer Hadway, Lise Desjardins, Ting-Yim Lee, Lisa Marie Hoffman, Diego Fraidenraich.

http://doi.org/10.1371/journal.pone.0174315

Abstract

Vascular endothelial growth factor (VEGF) and other pro-angiogenic growth factors have been investigated to enhance muscle tissue perfusion and repair in Duchenne muscular dystrophy (DMD). Current understanding is limited by a lack of functional data following in vivo delivery of these growth factors. We previously used dynamic contrast-enhanced computed tomography to monitor disease progression in murine models of DMD, but no study to date has utilized this imaging technique to assess vascular therapy in a preclinical model of DMD. In the current study, we locally delivered VEGF and ANG1 alone or in combination to dystrophic hind limb skeletal muscle. Using functional imaging, we found the combination treatment as well as ANG1 alone prevented decline in muscle perfusion whereas VEGF alone had no effect compared to controls. These findings were validated histologically as demonstrated by increased alpha-smooth muscle actin-positive vessels in muscles that received either VEGF+ANG1 or ANG1 alone compared to the sham group. We further show that ANG1 alone slows progression of fibrosis compared to either sham or VEGF treatment. The findings from this study shed new light on the functional effects of vascular therapy and suggest that ANG1 alone may be a candidate therapy in the treatment of DMD.

Partial Text

Vascular-targeted therapy to treat Duchenne muscular dystrophy (DMD) has been investigated since the early 2000’s [1]. The proposed mechanisms by which angiogenic therapy may alleviate the pathophysiology associated with DMD are numerous. Previous work has provided evidence of compromised vasculature in the disease, including impaired angiogenesis [2] and decreased vascular density in the mdx mouse, the most widely used murine model of DMD [3], as well as in the golden-retriever model of muscular dystrophy [4]. As such, many groups have attempted to increase vascular density in dystrophic muscle by treating it with vascular endothelial growth factor (VEGF), a well-known and potent inducer of angiogenesis [5–7]. Histological markers of endothelial cells, cells that make up the luminal wall of vessels, demonstrate increased vascular density following VEGF treatment. Other studies have also shown that VEGF levels are decreased in some muscle groups in mdx mice as well as in patients [8]. These findings are somewhat inconclusive, though, as others have shown the opposite: that VEGF levels are increased in dystrophic muscle tissue. This discrepancy is likely due to a temporally dependent alteration in expression that differs at various stages of the disease. Interestingly, hypoxia-inducible factor-1 alpha (HIF1-α) is increased in DMD patients [9] and others have shown that increases in HIF1-α in the mdx mouse brain correspond to elevated levels of VEGF [10].

Vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANG1) are increasingly being considered for their potential role in slowing disease progression in patients with Duchenne muscular dystrophy (DMD) [32]. While prior studies suggest a potential role for these factors in enhancing endogenous repair and cell therapy, studies remain to directly investigate the effects of either growth factor on functional perfusion in a non-invasive manner. Therefore, in the present study, we utilized the mdx/utrn+/- mouse, a murine DMD model more prone to fibrosis than the commonly used mdx mouse, to measure functional perfusion via dynamic contrast-enhanced computed tomography (DCE-CT). The short-term effect of VEGF, ANG1, or a combination of the two was assessed following a low dose, localized delivery for 16 days. Given the high degree of variability between animals with respect to baseline perfusion parameters, the ability to monitor vascular-targeted therapy over time in the same animal is particularly valuable. DCE-CT is a safe and effective means to monitor both disease progression and therapeutic efficacy and shows promise for scaling preclinical studies directly to patients [33,34]. Importantly, we provide the first evidence that VEGF alone is not sufficient to affect functional perfusion parameters in the hind limb skeletal muscle at the dose and duration investigated. Interestingly, we report here that ANG1 alone treatment is sufficient to affect functional perfusion, as demonstrated by a maintained blood volume compared to controls. To our knowledge, this is the first report of ANG1 alone having a significant, functional effect on perfusion in vivo in a murine model of DMD. While these findings are promising, they are not surprising given its role in promoting vascular maturity and satellite cell self-renewal [35]. As well, research in other vascular-related diseases such as cardiac ischemia and sepsis has uncovered the deleterious effect that low circulating levels of ANG1 may play in these states [36,37]. In human microvascular endothelial cells (HMVECs), serum from sepsis patients induced intercellular gap formation, and this effect was reversed by supplementation with ANG1 [38].

 

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http://doi.org/10.1371/journal.pone.0174315

 

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