Nitric oxide release combined with nonsteroidal antiinflammatory activity prevents muscular dystrophy pathology and enhances stem cell therapy

Abstract

Duchenne muscular dystrophy is a relatively common disease that affects skeletal muscle, leading to progressive paralysis and death. There is currently no resolutive therapy. We have developed a treatment in which we combined the effects of nitric oxide with nonsteroidal antiinflammatory activity by using HCT 1026, a nitric oxide-releasing derivative of flurbiprofen. Here, we report the results of long-term (1-year) oral treatment with HCT 1026 of two murine models for limb girdle and Duchenne muscular dystrophies (alpha-sarcoglycan-null and mdx mice). In both models, HCT 1026 significantly ameliorated the morphological, biochemical, and functional phenotype in the absence of secondary effects, efficiently slowing down disease progression. HCT 1026 acted by reducing inflammation, preventing muscle damage, and preserving the number and function of satellite cells. HCT 1026 was significantly more effective than the corticosteroid prednisolone, which was analyzed in parallel. As an additional beneficial effect, HCT 1026 enhanced the therapeutic efficacy of arterially delivered donor stem cells, by increasing 4-fold their ability to migrate and reconstitute muscle fibers. The therapeutic strategy we propose is not selective for a subset of mutations; it provides ground for immediate clinical experimentation with HCT 1026 alone, which is approved for use in humans; and it sets the stage for combined therapies with donor or autologous, genetically corrected stem cells.

Fig. 1.

Effects of treatment of α-SG-null mice with HCT 1026 or prednisolone on parameters of muscle function. (A) Creatine kinase serum levels. wt, wild-type. (B and C) Locomotor performance measured on the running-wheel (B) and exhaustion treadmill (C) tests. (D) Mechanical analysis of intact soleus and extensor digitorum longus (EDL) muscle contractile activity, assessed by measuring tetanic force. Values shown are the results of experiments on nine animals per group ±SEM. Asterisks and crosses indicate statistical significance vs. untreated α-SG-null mice analyzed in parallel as control (NT) or prednisolone-treated animals, respectively (P < 0.05).

Fig. 2.

Effects of treatment of α-SG-null mice with HCT 1026 or prednisolone on morphological muscle parameters. (A–C) Tibialis anterior and diaphragm muscle cross-cryosections were stained with hematoxylin/eosin (H&E). (A) Numbers of necrotic (n) and centronucleated (c) fibers. Values shown are the results of experiments on nine animals per group ± SEM. Asterisks indicate statistical significance vs. untreated α-SG-null mice analyzed in parallel as control (NT) or prednisolone-treated animals, respectively (P < 0.05). wt, wild-type. (B) Histological images obtained after 6 months of treatment representative of three reproducible experiments. (C) Fiber membrane damage was evaluated after the treadmill exhaustion test in EDL muscles at the 12-month time point by assessing Evans blue dye uptake. Shown are histological images representative of three reproducible experiments. (D) Distribution of CSA values of 300 single muscle fibers of tibialis anterior muscles isolated from nine animals per group. (Scale bar, 400 μm.)

Fig. 3.

Effects of treatment of α-SG-null mice with HCT 1026 or prednisolone on muscle inflammation. (A) Number of inflammatory infiltrates measured on Azan–Mallory-stained serial muscle sections of tibialis anterior (TA) and diaphragm (DIA) muscles. (B) TA muscles were isolated after 6 months of treatment and rapidly homogenized. The concentrations of the indicated cytokines were measured on muscle homogenates with appropriate antibodies. Values shown in A and B are the results of nine experiments ±SEM. Asterisks indicate statistical significance vs. untreated α-SG-null mice analyzed in parallel as control (NT) (P < 0.05). wt, wild-type. (C) Staining of TA muscle serial sections with Azan–Mallory reveals an accumulation of extracellular scar tissue (blue). (D) Presence of macrophages, revealed by staining of TA sections with the anti-CD11 and anti-laminin antibodies. (C and D) Histological images representative of nine reproducible experiments. [Scale bar (D), 400 μm.]

Fig. 4.

HCT 1026 increases the therapeutic efficacy of mesoangioblasts. α-SG-null mice, either untreated or exposed to a 3-month therapy with HCT 1026, were injected into the right femoral artery with GFP-expressing D16 mesoangioblasts treated or not with SDF-1. (A) Mesoangioblast migration to right (R) and left (L) gastrocnemius muscles (Gs) and liver (Li), lung (Lu), kidney (Ki), and spleen (Sp) evaluated by real-time PCR with specific primers for GFP on five animals per group. (B–E) Engrafting of mesoangioblasts to muscles. (B) Engrafting to the right and left quadriceps (Qd), Gs, and soleus (So) muscles evaluated by real-time PCR with specific primers for α-SG on three animals per group. (C) Western blot analysis of α-SG expression in Qd compared with that of GAPDH. The results shown are from one of three reproducible experiments. (D) Immunostaining of α-SG expressed in Qd fibers that were revealed in serial sections by staining with laminin; DAPI was used to identify nuclei. The results shown are from one of five reproducible experiments. (E) Specific tension of single muscle fibers (n = 101) of Gs from three animals per group. (F and G) Serum creatine kinase levels (F) and animal performance on the exhaustion treadmill tests (G), carried out as described in Fig. 1 on five animals per group. (E–G) Untreated indicates the values observed in α-SG-null mice which were neither treated with HCT 1026 nor injected with mesoangioblasts. (A, B, E–G) Bars represent SEM. Asterisks indicate statistical significance vs. α-SG-null mice injected with untreated mesoangioblasts analyzed in parallel as control (P < 0.05). wt, wild-type. [Scale bar (D), 200 μM.]