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

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.

Fig 1. VEGF and ANG1 are decreased in dystrophic diaphragm and gastrocnemius murine muscles.

ELISA analysis of VEGF and ANG1 in 9 to 10 week-old mdx/utrn+/- diaphragm and GM muscles compared to healthy wild type controls. A. VEGF was lower in mdx/utrn+/- diaphragm muscles compared to healthy wild-type controls. VEGF expression was not significantly different between dystrophic and healthy GM muscles. B. ANG1 was lower in mdx/utrn+/- diaphragm muscles compared to healthy wild-type controls. ANG1 expression was not significantly different between dystrophic and healthy GM muscles. n = 6 per group, *P < 0.05, by Student’s t-test. Error bars represent SD.

Fig 2. Perfusion measured at endpoint is not significantly different between hind limbs, regardless of treatment.

(A) Schematic representation of treatment groups. “Sham” group mice received sham injections in both hind limbs. “VEGF” mice received sham injection in the right hind-limb, VEGF in the contralateral limb. “VEGF+ANG1” mice received VEGF in the right hind-limb and VEGF+ANG1 in the contralateral limb. “ANG1” mice received ANG1 in the left hind limb and a sham injection in the contralateral limb. (B) Blood flow (left) and blood volume (right) did not differ between hind limbs, allowing for perfusion measurements to be assessed based on the averaged BF and BV following treatment. n = 6, P < 0.05, by Wilcoxan signed-rank test.

Fig 3. VEGF+ANG1 slows decline in hind limb muscle perfusion blood flow 16 days post-treatment in mdx/utrn+/- mice.

A. Representative DCE-CT blood flow maps of sham-injected, VEGF-, VEGF+ANG1-, and ANG1-treated hind limbs. B. Blood flow decreased in all but two mice over the course of the study. n = 6, P < 0.05, by one-way ANOVA. Error bars represent SD. Means with different letters are significantly different.

Fig 4. Both VEGF+ANG1 and ANG1 treatment prevent decline in blood volume 16 days post-treatment in mdx/utrn+/- hind limb skeletal muscle.

A. Representative DCE-CT blood volume maps of sham-injected, VEGF-, VEGF+ANG1-, and ANG1-treated hind limbs. B. Blood volume was not significantly different in sham-injected and VEGF-treated hind limbs. VEGF+ANG1 treatment resulted in significantly higher fold-change in blood volume compared to both VEGF and sham group. n = 6, P < 0.05, by one-way ANOVA. Error bars represent SD. Means with different letters are significantly different.

Fig 5. VEGF and ANG1 increase vascular density following localized delivery in the gastrocnemius muscle of mdx/utrn+/- mice.

A. Representative images of CD31 expression (green) in gastrocnemius muscles of mdx/utrn+/- following 16 days of angiogenic growth factor treatment. DAPI was used as a counterstain (blue). Scale bar = 100μm. B. Quantification of CD31 expression in the four treatment groups. n = 4, p<0.05 by one-way ANOVA. Error bars represent ± SD and means with different letters are significantly different.

Fig 6. ANG1 treatment increases vessel maturation following treatment in mdx/utrn+/- GM muscle.

A. Representative immunofluorescence images of αSMA expression (green) in sham-injected, VEGF- and VEGF+ANG1-treated GM muscles. B. Immunohistochemical analysis of αSMA-positive vessels, represented as percent image area. n = 4 for all groups, by one-way ANOVA. Error bars represent SD and means with different letters are significantly different. C. Growth factor-coated beads are visible at the injection site. αSMA-positive vessels are detected at the injection site of VEGF+ANG1- and ANG1-treated hind limbs, and absent at the injection site of sham-injected and VEGF-treated hind limbs. DAPI was used as a counterstain. Scale bar = 100 μm.

Fig 7. ANG1 treatment decreases collagen deposition following treatment in mdx/utrn+/- GM muscle.

A. Representative Masson’s trichrome-stained tissue sections of sham-injected, VEGF-, VEGF+ANG1, and ANG1-treated GM muscles. Collagen deposition appears blue. Scale bar = 100 μm B. Quantification of collagen deposition represented as percent image area. n = 4 for all groups P < 0.05 by one-way ANOVA. Error bars represent SD and means with different letters are significantly different. Scale bar = 100 μm.