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.
- SourceAvailable from: Clara De Palma[show abstract] [hide abstract]
ABSTRACT: The mechanism of skeletal myoblast fusion is not well understood. We show that endogenous nitric oxide (NO) generation is required for myoblast fusion both in embryonic myoblasts and in satellite cells. The effect of NO is concentration and time dependent, being evident only at the onset of differentiation, and direct on the fusion process itself. The action of NO is mediated through a tightly regulated activation of guanylate cyclase and generation of cyclic guanosine monophosphate (cGMP), so much so that deregulation of cGMP signaling leads to a fusion-induced hypertrophy of satellite-derived myotubes and embryonic muscles, and to the acquisition of fusion competence by myogenic precursors in the presomitic mesoderm. NO and cGMP induce expression of follistatin, and this secreted protein mediates their action in myogenesis. These results establish a hitherto unappreciated role of NO and cGMP in regulating myoblast fusion and elucidate their mechanism of action, providing a direct link with follistatin, which is a key player in myogenesis.The Journal of Cell Biology 02/2006; 172(2):233-44. · 10.82 Impact Factor
- The Lancet Neurology 06/2005; 4(5):264. · 23.92 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Objective: Nitric oxide–donating nonsteroidal anti-inflammatory drugs (NO-NSAIDs) are a new class of cyclooxygenase (COX) inhibitors. To investigate whether these drugs actually release nitric oxide (NO), we labeled the nitroxy group of nitroflurbiprofen with nitrogen 15 to determine the metabolic fate of this compound in humans.Method: Six healthy volunteers who fasted were given an oral dose of 15N-nitroflurbiprofen (100 mg). Samples of blood, urine, and gastric headspace gas were taken over a 24-hour period to determine the levels of nitroflurbiprofen, flurbiprofen, total nitrate/nitrite, 15N-nitrate/nitrite, COX activity, and gastric NO. In a crossover study (1 week apart), a further 6 healthy volunteers who fasted were given an oral dose of nitroflurbiprofen (100 mg) or flurbiprofen (65 mg) and levels of gastric NO were determined.Results: Nitroflurbiprofen was undetectable in the systemic circulation. Levels of 15N-nitrate/nitrite (5.2% 1.5% enrichment) and flurbiprofen (2.4 0.7 g/mL) peaked at 4 hours in the plasma and gradually decreased thereafter. In unstimulated blood, the plasma levels of thromboxane B2 (COX-1 activity) were 2 to 3 ng/mL, and after calcium ionophore stimulation, large amounts of thromboxane B2 were produced (112 31 ng/mL). Prostaglandin E2 was undetectable in unstimulated blood. After lipopolysaccharide stimulation, the plasma levels of prostaglandin E2 increased to 15 4 ng/mL. The metabolite flurbiprofen inhibited plasma COX-1 activity for the duration of the study period (maximum inhibition at 4 hours), whereas COX-2 activity recovered after 6 hours. In the crossover study, levels of gastric NO were higher in subjects given nitroflurbiprofen, when compared with those given flurbiprofen. (The area under the curve for gastric NO was 435 107 ppm h versus 305 94 ppm h [95% confidence interval of the difference, 89-172 ppm h; P < .001]).Conclusion: Nitroflurbiprofen was undetectable in the systemic circulation, suggesting metabolism to 15N-nitrate/nitrite and flurbiprofen in the presystemic circulation. Levels of gastric NO were significantly higher after ingestion of nitroflurbiprofen than flurbiprofen.Clinical Pharmacology & Therapeutics 09/2004; 76(4):350-358. · 6.85 Impact Factor
Nitric oxide release combined with nonsteroidal
antiinflammatory activity prevents muscular
dystrophy pathology and enhances stem cell therapy
Silvia Brunelli*†, Clara Sciorati†, Giuseppe D’Antona‡, Anna Innocenzi†, Diego Covarello†, Beatriz G. Galvez†,
Cristiana Perrotta†§, Angela Monopoli¶, Francesca Sanvito†, Roberto Bottinelli‡, Ennio Ongini¶, Giulio Cossu†?**,
and Emilio Clementi†§**††
*Department of Experimental Medicine, University of Milano–Bicocca, 20052 Monza, Italy;†San Raffaele Scientific Institute, Stem Cell Research Institute,
Via Olgettina 58, 20132 Milan, Italy;‡Department of Experimental Medicine, University of Pavia, 27100 Pavia, Italy;¶Nicox Research Institute,
Via Ariosto 21, 20091 Bresso, Italy;?Department of Biology, University of Milano, 20130 Milan, Italy;††E. Medea Scientific Institute,
23842 Bosisio Parini, Italy; and§Department of Preclinical Sciences, University of Milano, 20157 Milan, Italy
Edited by Salvador Moncada, University of London, London, United Kingdom, and approved November 13, 2006 (received for review September 20, 2006)
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
(?-sarcoglycan-null and mdx mice). In both models, HCT 1026
significantly ameliorated the morphological, biochemical, and
functional phenotype in the absence of secondary effects, effi-
ciently slowing down disease progression. HCT 1026 acted by
reducing inflammation, preventing muscle damage, and preserv-
ing 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
HCT 1026 ? satellite cells ? skeletal muscle ? ?-sarcoglycan-null mice ?
for which there is no resolutive therapy (1). In the most severe
forms, such as Duchenne muscular dystrophy, muscle regeneration
and death, usually by respiratory and/or cardiac failure (1). The
therapeutic protocols currently in use, based on corticosteroid
administration, provide some delay in the progression of the
disease, but they are associated with severe side effects (2). The
favorable outcomes in clinical trials, and they have not entered into
the clinical practice (3).
Studies in mouse models have explored possible therapeutic
strategies, ranging from deacetylase inhibitors (4), inhibition of
myostatin (5), insulin-like growth factor 1 overexpression (6) to
skipping of the mutated exon (7) and cell therapy with mesoan-
lack approval for use in patients and data on long-term efficacy,
safety, and tolerability; in addition, they are expensive and in some
cases they target only subsets of patients. The development of stem
uscular dystrophies are clinically and molecularly heteroge-
neous genetic diseases causing wasting of skeletal muscle,
cell therapies is also hampered by problems related with isolation,
in vitro expansion, and efficient engraftment of the cells used (9).
To tackle these issues, we developed a treatment by combining
and regeneration (10–15) with nonsteroidal antiinflammatory
activity. As a drug of choice, we selected HCT 1026, a derivative
of flurbiprofen, one of the most potent nonsteroidal antiinflam-
matory drugs, that releases NO and does not have the severe side
effects of corticosteroids (16, 17). Of importance for an imme-
diate testing in the clinic is the fact that HCT 1026 is safe at the
gastrointestinal level, and it has been approved for use in
humans; it is effective on oral administration, and it is thus suited
for long-term treatment. As a second important step in the
development of an efficacious therapeutic protocol for muscular
dystrophy, we combined this pharmacological treatment with
intraarterial delivery of mesoangioblasts.
The effect of HCT 1026 was tested on two models of muscular
dystrophy, the ?-sarcoglycan (SG)-null and mdx mice, in a long-
term (1-year) treatment. HCT 1026 dramatically slowed the
progress of the disease, and it maintained muscle integrity and
function through mechanisms involving inhibition of inflammation
significantly more potent than prednisolone, which was used as a
reference corticosteroid, and it did not induce detectable side
effects. Moreover, the drug treatment improved significantly the
to dystrophic muscles. These results open a window to an effective
cure of muscular dystrophy.
The first set of experiments was carried out in the ?-SG-null mice,
a mouse model of limb girdle muscular dystrophy with a severe
phenotype (18). Groups of mice (18 animals per group) were
treated for up to 12 months with HCT 1026 (30 mg/kg of body
Author contributions: S.B. and C.S. contributed equally to this work; S.B., A.M., R.B., E.O.,
G.C., and E.C. designed research; S.B., C.S., G.D., A.I., D.C., B.G.G., C.P., and F.S. performed
research; A.M. and E.O. contributed new reagents/analytic tools; S.B., C.S., G.D., R.B., E.O.,
G.C., and E.C. analyzed data; and S.B., E.O., G.C., and E.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
Abbreviations: CSA, cross-sectional area; EDL, extensor digitorum longus; ISDN, isosorbide
protein 1?; NO, nitric oxide; SDF-1, stromal cell-derived factor 1; SG, sarcoglycan.
**To whom correspondence may be addressed at: Stem Cell Research Institute, H. San
Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy. E-mail: giulio.
firstname.lastname@example.org or email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2006 by The National Academy of Sciences of the USA
January 2, 2007 ?
vol. 104 ?
no. 1 www.pnas.org?cgi?doi?10.1073?pnas.0608277104
groups receiving the same diet without any drug or the NO donor
isosorbide dinitrate (ISDN; 30 mg/kg) were analyzed in parallel.
Flurbiprofen was not used because of its known long-term toxicity
(16, 17), whereas the daily dose of HCT 1026 we used produces
antiinflammatory activity without signs of toxicity (19). Incorpo-
ration of the drug into the diet led to plasma levels of flurbiprofen
(the active and detectable metabolite of HCT 1026) in the range of
16–20 ?M after 30 days or 44 ?M after 6 months (19). This plasma
concentration is in the same order of magnitude of that found in
healthy volunteers after treatment for 7 days with HCT 1026 at 100
after weaning, roughly at the onset of the disease symptoms, thus
mimicking a treatment starting in pediatric patients. To evaluate
efficacy, we relied on morphological, histochemical, and functional
analyses of bone, liver, spleen, brain, kidney, stomach, lung, and
histological differences in these organs were observed between
?-SG-null and wild-type (WT) mice, and no significant histological
alterations were observed in the 12-month time window of drug
HCT 1026 Reduces Creatine Kinase Serum Levels in ?-SG-Null Mice.
is commonly used as a biomarker of the severity of muscular
dystrophies. The serum levels of creatine kinase in untreated
?-SG-null mice were higher than those of WT animals already
within the first 2 months of life, and they increased progressively up
with the dramatic reduction in muscular tissue known to occur at
later stages of the disease (18) (see also Fig. 2). In the group
receiving HCT 1026, creatine kinase levels were reduced signifi-
cantly with respect to those observed in untreated animals (by
22.2%, 44.9%, and 75.7% at 3, 4, and 6 months, respectively; P ?
in prednisolone-treated animals (by 41.0%, 42.2%, and 53.6% at 3,
4, and 6 months, respectively P ? 0.05; n ? 9) (Fig. 1A), whereas
ISDN did not have significant effects (SI Table 1).
Mice. Muscle function was evaluated by using the running-wheel
and treadmill tests, which measure animal voluntary motility and
muscle endurance, respectively. Untreated ?-SG-null mice showed
a significantly reduced motility in the running-wheel test (Fig. 1B)
and a lower time of exhaustion in the treadmill test (Fig. 1C)
compared with WT mice. The groups receiving HCT 1026 per-
formed significantly better than untreated dystrophic mice in both
tests at all time points. At 6 months, the group receiving pred-
nisolone performed significantly worse than the group receiving
HCT 1026 on the running wheel, and they performed at compa-
rable levels on the treadmill. At 12 months, prednisolone-treated
HCT 1026. Prednisolone-treated mice performed significantly bet-
group receiving ISDN behaved similarly to untreated ?-SG-null
mice (SI Table 1).
We determined whether the reduced creatine kinase serum
levels and the increase in the in vivo performance induced by the
treatments correlated with increased muscle strength. To this end,
at 12 months we measured force production by soleus and EDL
muscles. Muscles from ?-SG-null mice developed less force during
tetanic contraction compared with WT mice (Fig. 1D). Treatment
with HCT 1026 reduced this difference significantly. Prednisolone
was significantly less effective in preventing the loss of muscle force
in soleus muscle, and it did not show any effect in EDL. At the end
by evaluating the uptake of Evans blue dye, which stains severely
damaged and dying fibers. EDL and soleus from ?-SG-null mice,
uptake compared with EDL from WT mice (Fig. 2C). No signif-
icant differences between these groups could be detected. By
contrast, muscles from ?-SG-null mice treated with HCT 1026
displayed a significantly lower dye uptake (Fig. 2C and data not
HCT 1026 Enhances Regeneration and Protects Skeletal Muscle in
?-SG-Null Mice from Damage. Evaluation of H&E-stained sections
was carried out on the tibialis anterior and diaphragm muscles. The
muscle obtained from untreated ?-SG-null mice revealed a pro-
fibers, which was accompanied by an increase in centronucleated,
regenerating fibers early on, followed by a decrease, most likely
because of depletion of satellite cells (22) (Fig. 2 A and B). The
eters, whereas ISDN produced no significant effects (SI Table 1).
By contrast, muscle architecture was nearly normal in the groups
receiving HCT 1026, with a significantly lower number of necrotic
fibers, even at the 12-month time point. Interestingly, the number
of centronucleated fibers was higher in HCT 1026-treated animals,
and it did not decline with time, indicating that exhaustion of the
satellite pool did not occur, consistent with the creatine kinase
serum level determinations and the observed reduction in muscle
HCT 1026 came from the analyses of frequency histograms of the
cross-sectional area (CSA) of the single fibers. In the untreated
?-SG-null mice, fiber CSA was very heterogeneous compared with
fiber CSA of WT mice, with values distributed between 250 and
3,000 ?m2, the largest diameters being a sign of degeneration
(pathological hypertrophy) (Fig. 2D). By contrast, the frequency
histograms of HCT 1026-treated animals showed a significantly
more homogeneous distribution, with CSA values not significantly
different from those observed in the WT mice. Homogeneity in
CSA values was observed also in prednisolone-treated animals, but
on parameters of muscle function. (A) Creatine kinase serum levels. wt,
(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
ne-treated animals, respectively (P ? 0.05).
Brunelli et al.PNAS ?
January 2, 2007 ?
vol. 104 ?
no. 1 ?
only up to 6 months; after 12 months CSA value distribution was as
heterogeneous as that of untreated ?-SG-null mice.
HCT 1026 Increases Satellite Cell Differentiation and Resistance to
Apoptosis, and It Preserves Their Number in Vivo. The results
obtained so far suggest that the mechanism of action of HCT
1026 involves specific beneficial effects on satellite cells. We
therefore assessed the action of the drug on satellite cell
differentiation, proliferation, and resistance to apoptogenic cues
found in the cytotoxic environment of the dystrophic muscle.
The results obtained by both in vitro and in vivo experiments
clearly show that treatment with HCT 1026 significantly in-
creased satellite cell differentiation and survival, leading to an
increased satellite cell population (SI Results and SI Fig. 6). The
efficacy of HCT 1026 on these parameters was still significant
after 12 months of treatment.
HCT 1026 and Prednisolone Exert an Antiinflammatory Action on
Skeletal Muscle of ?-SG-Null Mice. Inflammation plays a role in the
pathogenesis of muscular dystrophy because an abundant inflam-
dystrophic muscle progressively replaces myofibers, causing reduc-
tion of muscle strength and worsening the disease (23). Azan–
Mallory staining revealed frequent foci of fibrosis in muscles of
untreated ?-SG-null mice (Fig. 3 A and B), with an infiltrate
revealed by laminin staining (Fig. 3D). We examined also the
expression of several proinflammatory cytokines, and we estab-
protein-1? (MIP-1?), and monocyte chemoattractant protein-1
(MCP-1) are increased (Fig. 3C). We found that treatment with
HCT 1026 significantly reduced the number of inflammatory
infiltrates, the amount of fibrotic tissue, and the concentrations of
TGF-?, MIP-1?, and MCP-1, indicating that inhibition of inflam-
reduced the inflammatory infiltrates and cytokine expression;
however, it did not reduce the formation of fibrous tissue. These
effects, together with the limited effect on long-term muscle
regeneration and protection from damage (Fig. 2), may contribute
an explanation of why prednisolone does not significantly amelio-
rate muscle function.
HCT 1026 Ameliorates the Dystrophic Phenotype of the mdx Mouse.
mdx mice (nine animals per group) were treated with HCT 1026
(30 mg/kg) incorporated in the diet, or they received plain diet
wheel and treadmill tests, and muscle morphology were assessed
after 6 months. The results clearly show that the long-term
treatment with HCT 1026 exerts beneficial effects also in these
1026 significantly reduced creatine kinase levels, improved mo-
tility tests, reduced damage to the muscles, and increased
centronucleated fibers. Also, in the mdx mouse we found no
signs of toxicity with HCT 1026 in a variety of tissues and organs
(data not shown).
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 ?-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
of tibialis anterior muscles isolated from nine animals per group. (Scale bar, 400 ?m.)
www.pnas.org?cgi?doi?10.1073?pnas.0608277104 Brunelli et al.
HCT 1026 Enhances the Therapeutic Efficacy of Mesoangioblasts by
Increasing Their Ability to Migrate and Engraft to Dystrophic Muscles.
The treatment with HCT 1026 does not correct the genetic defect
underlying muscular dystrophy. To address this issue we combined
the pharmacological treatment with delivery of mesoangioblasts.
GFP (8) to trace them into the various tissues, and they were used
either untreated or after a 12-h ex vivo exposure to stromal
cell-derived factor 1 (SDF-1; 50 ng/ml), a treatment that increases
their homing to muscle (24). At variance with a previous report in
performed a single injection to evaluate best the changes in
therapeutic efficacy induced by drug treatment. Migration of
mesoangioblasts was assessed by injecting them at a dose of 5 ? 105
with HCT 1026. Gastrocnemius and filter organs (spleen, liver,
lung, and kidney, where mesoangioblasts that flow through the
circulation may be trapped) were recovered 6 h after the injection,
and the percentage of the cells migrated in them was evaluated by
real-time PCR with specific primers for GFP. Mesoangioblast
migration in gastrocnemius on the side of injection (right side) was
significantly enhanced in HCT 1026-treated animals (Fig. 4A). A
synergic effect of HCT 1026 with pretreatment of mesoangioblasts
with SDF-1 before their injection was also observed. The percent-
age of cells retained by the filter organs was reduced in all
conditions favoring mesoangioblast migration to the muscle.
To test whether an increased migration would lead also to an
or without HCT 1026 were killed 2 months after mesoangioblast
injection. The efficacy of the treatment was assessed by measuring
?-SG mRNA expression by RT-PCR and ?-SG protein levels by
Western blotting and immunofluorescence. Mesoangioblasts en-
grafted and reconstituted muscle fibers in the quadriceps, gastroc-
nemius, and soleus muscles on the side of injection (Fig. 4 B–D).
This effect was doubled in HCT 1026-treated animals and also by
preexposure of mesoangioblasts to SDF-1. The combination of
these two treatments increased 4-fold the percentage of fibers
engrafting in the contralateral muscles also. The ability of HCT
1026 to increase mesoangioblast-mediated fiber reconstitution was
accompanied by increased single fiber-specific tension (Fig. 4E),
reduced muscle damage (judged by reduced creatine kinase levels;
Fig. 4F), and enhanced mouse performance on the treadmill (Fig.
4G), which were significant vs. those observed in animals receiving
mesoangioblasts in the absence of HCT 1026 treatment, and they
were further increased by SDF-1 pretreatment of mesoangioblasts.
Thus, HCT 1026 greatly increases mesoangioblast engrafting, and
it may synergize with other treatments, leading to a significantly
more effective cell therapy.
Two decades after the identification of the molecular defect re-
sponsible for Duchenne muscular dystrophy, there are still no
effective cures for the disease. The failure of all previous pharma-
cological treatments has left corticosteroids as the only available
drug treatment. Therapy with corticosteroids, despite current at-
tempts to ameliorate the protocols of administration, is still of
limited clinical benefits, and it is accompanied by severe side ef-
The treatment we tested here meets several criteria for an
effective therapy, including the ability to block or at least signifi-
cantly delay the progress of the disease, produce little or no side
toxicity, be cost-effective, and, eventually, resolve the underlying
genetic defect. In particular, the administration of HCT 1026 was
sufficient on its own to delay significantly and persistently the
progression of muscular dystrophy in two different models relevant
to muscular dystrophy in humans. The drug was effective in
correcting biochemical and morphological alterations and in lim-
iting inflammation. Most importantly, the drug increased muscle
strength and significantly increased the ability of animals to per-
form on different motility tests. This functional amelioration was
persistent after 12 months of treatment, when disease in untreated
animal was severe, clearly demonstrating the efficacy of the treat-
ment in slowing disease progression. Long-term beneficial effects
have not been reported for any of the experimental treatments of
muscular dystrophy investigated so far.
The beneficial effects of NO on muscle function are well known,
NO is generated by skeletal muscle to stimulate key actions in
muscle repair, including activation of satellite cells and release of
myotrophic factors (10–12). In addition, NO stimulates vasodila-
tion, and thus increases the supply of oxygen and glucose uptake,
and it triggers mitochondrial biogenesis, all of which contribute to
preserve muscle from damage during contraction (13, 14, 25, 26).
Indeed, amelioration of the dystrophic phenotype had been ob-
stimulated investigations about the therapeutic potential of treat-
of NO, or molsidomine, a NO donor, to increase NO release (26,
27). Although some amelioration of muscle morphology was ob-
served, and in one study creatine kinase levels were reduced (26),
were short-term investigations. We found no amelioration of mor-
phofunctional parameters in ?-SG-null mice after a 6-month treat-
ment with ISDN.
The detectable and persistent recovery of muscle function ob-
served in our study is most likely because HCT 1026 combines the
Azan–Mallory-stained serial muscle sections of tibialis anterior (TA) and dia-
phragm (DIA) muscles. (B) TA muscles were isolated after 6 months of treat-
were measured on muscle homogenates with appropriate antibodies. Values
statistical significance vs. untreated ?-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 repre-
sentative of nine reproducible experiments. [Scale bar (D), 400 ?m.]
Brunelli et al.PNAS ?
January 2, 2007 ?
vol. 104 ?
no. 1 ?
multiple beneficial effects of NO outlined above with the potent
antiinflammatory action of flurbiprofen, yielding a drug endowed
with new properties. We found that important mechanisms concur
to determine the excellent therapeutic potential of HCT 1026. The
drug inhibited significantly inflammation in the dystrophic muscle
by reducing the generation of proinflammatory cytokines and
reducing infiltration of proinflammatory cells. In addition, HCT
1026 had a specific action in preserving satellite cell number and
function by protecting them from the proapoptotic environment of
This action explains why in HCT 1026-treated animals, the regen-
erating ability of muscle, assessed as an increased number of
centronucleated, reduced number of necrotic fibers and normal
CSA, was preserved. The beneficial effect of the treatment we
propose was tested against prednisolone, one of the widely used
found that HCT 1026 was more effective than the steroid in
ameliorating morphological, biochemical, and functional parame-
ters and that these actions occurred in the absence of toxic side
effects. In particular, we did not observe gastric damage, which in
HCT 1026 is minimized by the NO-donating moiety (16). More-
over, the limited beneficial effects of the steroid decreased with
time, becoming insignificant at later stages of the disease.
Previous studies in the mdx mouse showing that corticosteroids
either alone or combined with other treatments exert beneficial
effects were designed only as short-term therapeutic protocols (2–3
months on average) (25, 28). In humans, however, treatments have
to be delivered long-term, a situation in which the side effects of
corticosteroid administration still represent a serious problem (21).
In contrast, HCT 1026 has a profile of safety that holds promise for
appears a more promising therapy than the combination of L-
arginine and deflazacort recently proposed (25). In addition, L-
arginine and deflazacort were tested only up to 3 months, and
dramatic decrease of NO synthase observed in dystrophic muscles
(13). The second important advantage of the treatment with HCT
1026 is that it significantly enhanced the engrafting to muscle of
to be effective in correcting the genetic defect of the muscle (8).
This enhancement resulted in a significant increase in their thera-
peutic efficacy as shown by morphological and biochemical param-
eters and by in vivo motility assay on the treadmill. The effect was
further enhanced by in vitro exposure of donor cells to SDF-1, a
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
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 ?-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. ?-SG-null mice injected with untreated mesoangioblasts analyzed
in parallel as control (P ? 0.05). wt, wild-type. [Scale bar (D), 200 ?M.]
www.pnas.org?cgi?doi?10.1073?pnas.0608277104Brunelli et al.
that HCT 1026 may be used in combined therapeutic strategies to
yield synergic beneficial effects. Of importance, the increased
homing of mesoangioblasts to the muscle was accompanied by a
This number is large, and it may cause damage to these organs (9).
An increased efficiency of stem cell delivery will reduce either the
number of cells needed for a single injection or the number of
injections, leading to an optimization of cell therapy.
Compared with the current experimental therapies, the treat-
ment we propose has clear advantages. Despite recent encouraging
results, therapy with stem cells alone cannot reverse the preexisting
pathological changes, and it still suffers from inefficient engraft-
ment (9). This inefficiency may originate from a limited homing of
these cells to muscle and a reduced ability to resist to the cytotoxic
environment existing in the damaged muscle (23, 29). These
problems are reduced by the combination of mesoangioblasts with
Within the current scenario of preclinical and early clinical
studies, HCT 1026 shows the distinct advantages of being econom-
ically affordable (unlike humanized anti-myostatin antibodies),
nonimmunogenic (like adeno-associated vectors), suitable for all
mutations (unlike oligonucleotides for exon skipping), and with a
tested profile of safety. We conclude that the therapeutic strategy
presented in this work represents a significant advance and sets the
stage for immediate trials in patients.
Materials and Methods
Animal Treatments. Mice were treated with HCT 1026 and pred-
nisolone compounded in the chow following the European Com-
Committee. Mesoangioblast D16-GFP cells, pretreated with or
without SDF-1, were delivery by intraarterial injection through the
right femoral artery (8).
Determination of Creatine Kinase Serum Levels. Serum creatine
kinase concentrations were measured from mouse blood samples
by using the indirect colorimetric assay (Roche Diagnostics, Basel,
Switzerland), following standard procedures.
Functional Tests. For a description of the functional tests, see SI
Materials and Methods.
Mechanics of Isolated Muscles and Fibers. Mechanical analysis of
intact muscles was carried out as described in ref. 30. Tetanic
force was normalized to the estimated CSA assuming cylindrical
shape of the muscle and a density of 1.06 mg/mm3(CSA
corresponds to the wet weight divided by its length). Normalized
tetanic force is expressed as kN/m2. Absolute (Po) and specific
force (Po/CSA) of single muscle fibers were measured by using
skinned preparations (31).
Histology Immunohistochemistry and Morphometry. Muscles were
dissected and frozen in liquid N2-cooled isopentane. Fiber mem-
sections were stained in H&E, by using the Azan–Mallory tech-
nique, or immunostained as described in ref. 8. Morphometrical
analyses to evaluate distribution of fiber CSA were carried out on
300 fibers per muscle, on H&E-stained sections of tibialis anterior,
by using Image 1.63 software (Scion Corporation, Frederick, MD).
Cytokine Analysis. TGF-? and MIP-1? concentrations were deter-
mined on equal amounts of protein lysates from tibialis anterior
muscles, by using the Quantikine ELISA kits (R&D Systems,
Minneapolis, MN). MCP-1 concentration was assessed by flow
cytometry by using the cytometric bead array (BD Bioscience, San
RNAs and Protein Measurements. Real-time quantitative PCR anal-
ysis was carried out on cDNA from tissue samples by using an
Mx3000P real-time PCR detection system (Stratagene, La Jolla,
CA). Each cDNA sample was amplified in duplicate by using the
GFP: forward, AAGTTCATCTGCACCACCG; reverse, TCCT-
tissue were analyzed by Western blotting by using mAbs anti-?-SG
(Novocastra, Newcastle upon Tyne, U.K.) and GAPDH (Biogen-
esis, Kingston, NH) (8).
Statistical Analyses. Values were expressed as means ?SEM. Sta-
tistical significance was assessed by one-way ANOVA followed by
the Student–Newman–Keul test. A probability of ?5% (P ? 0.05)
was considered statistically significant.
We thank K. Campbell for the ?-SG-null mice, M. Sampaolesi for advice,
and C. Rinaldi for technical help. This work was supported by Telethon
Grant GGP05007 (to E.C.), Association Franc ¸aise contre les Myopathies
Grant 12048 (to E.C.), The European Community [Euro Stem Cell (to
G.C.)], Associazione Parent Project Italia (to G.C.), the Italian Association
of Cancer Research Grant 1016 (to E.C.), and the Ministry of Health
[Ricerca Corrente (to E.C.)].
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