Differential Effects of Halofuginone Enantiomers on Muscle Fibrosis and Histopathology in Duchenne Muscular Dystrophy

Progressive loss of muscle and muscle function is associated with significant fibrosis in Duchenne muscular dystrophy (DMD) patients. Halofuginone, an analog of febrifugine, prevents fibrosis in various animal models, including those of muscular dystrophies. Effects of (+)/(−)-halofuginone enantiomers on motor coordination and diaphragm histopathology in mdx mice, the mouse model for DMD, were examined. Four-week-old male mice were treated with racemic halofuginone, or its separate enantiomers, for 10 weeks. Controls were treated with saline. Racemic halofuginone-treated mice demonstrated better motor coordination and balance than controls. However, (+)-halofuginone surpassed the racemic form’s effect. No effect was observed for (−)-halofuginone, which behaved like the control. A significant reduction in collagen content and degenerative areas, and an increase in utrophin levels were observed in diaphragms of mice treated with racemic halofuginone. Again, (+)-halofuginone was more effective than the racemic form, whereas (−)-halofuginone had no effect. Both racemic and (+)-halofuginone increased diaphragm myofiber diameters, with no effect for (−)-halofuginone. No effects were observed for any of the compounds tested in an in-vitro cell viability assay. These results, demonstrating a differential effect of the halofuginone enantiomers and superiority of (+)-halofuginone, are of great importance for future use of (+)-halofuginone as a DMD antifibrotic therapy.

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Targeting fibrosis in the Duchenne Muscular Dystrophy mice model: an uphill battle

10.1101/2021.01.20.427485 ◽

2021 ◽

Author(s):

Marine Theret ◽

Marcela Low ◽

Lucas Rempel ◽

Fang Fang Li ◽

Lin Wei Tung ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Large Scale ◽

Drug Screen ◽

Mdx Mice ◽

Mice Model ◽

Positive Effects ◽

In Vitro Drug Screening

AbstractAimFibrosis is the most common complication from chronic diseases, and yet no therapy capable of mitigating its effects is available. Our goal is to unveil specific signallings regulating the fibrogenic process and to identify potential small molecule candidates that block fibrogenic differentiation of fibro/adipogenic progenitors.MethodWe performed a large-scale drug screen using muscle-resident fibro/adipogenic progenitors from a mouse model expressing EGFP under the Collagen1a1 promotor. We first confirmed that the EGFP was expressed in response to TGFβ1 stimulation in vitro. Then we treated cells with TGFβ1 alone or with drugs from two libraries of known compounds. The drugs ability to block the fibrogenic differentiation was quantified by imaging and flow cytometry. From a two-rounds screening, positive hits were tested in vivo in the mice model for the Duchenne muscular dystrophy (mdx mice). The histopathology of the muscles was assessed with picrosirius red (fibrosis) and laminin staining (myofiber size).Key findingsFrom the in vitro drug screening, we identified 21 drugs and tested 3 in vivo on the mdx mice. None of the three drugs significantly improved muscle histopathology.SignificanceThe in vitro drug screen identified various efficient compounds, none of them strongly inhibited fibrosis in skeletal muscle of mdx mice. To explain these observations, we hypothesize that in Duchenne Muscular Dystrophy, in which fibrosis is a secondary event due to chronic degeneration and inflammation, the drugs tested could have adverse effect on regeneration or inflammation, balancing off any positive effects and leading to the absence of significant results.

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G.P.7.07 In vitro dissection of the pathogenic mechanisms of muscle fibrosis in Duchenne muscular dystrophy

Neuromuscular Disorders ◽

10.1016/j.nmd.2008.06.187 ◽

2008 ◽

Vol 18 (9-10) ◽

pp. 778

Author(s):

S. Zanotti ◽

S. Saredi ◽

C. Cappelletti ◽

A. Ruggieri ◽

F. Blasevich ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Pathogenic Mechanisms ◽

Muscle Fibrosis

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Menstrual Blood-derived Cells Confer Human Dystrophin Expression in the Murine Model of Duchenne Muscular Dystrophy via Cell Fusion and Myogenic Transdifferentiation

Molecular Biology of the Cell ◽

10.1091/mbc.e06-09-0872 ◽

2007 ◽

Vol 18 (5) ◽

pp. 1586-1594 ◽

Cited By ~ 120

Author(s):

Chang-Hao Cui ◽

Taro Uyama ◽

Kenji Miyado ◽

Masanori Terai ◽

Satoru Kyo ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Progenitor Cells ◽

Cell Fusion ◽

Menstrual Blood ◽

Mdx Mice ◽

Dystrophin Expression ◽

Human Dystrophin

Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder in children, is an X-linked recessive muscle disease characterized by the absence of dystrophin at the sarcolemma of muscle fibers. We examined a putative endometrial progenitor obtained from endometrial tissue samples to determine whether these cells repair muscular degeneration in a murine mdx model of DMD. Implanted cells conferred human dystrophin in degenerated muscle of immunodeficient mdx mice. We then examined menstrual blood–derived cells to determine whether primarily cultured nontransformed cells also repair dystrophied muscle. In vivo transfer of menstrual blood–derived cells into dystrophic muscles of immunodeficient mdx mice restored sarcolemmal expression of dystrophin. Labeling of implanted cells with enhanced green fluorescent protein and differential staining of human and murine nuclei suggest that human dystrophin expression is due to cell fusion between host myocytes and implanted cells. In vitro analysis revealed that endometrial progenitor cells and menstrual blood–derived cells can efficiently transdifferentiate into myoblasts/myocytes, fuse to C2C12 murine myoblasts by in vitro coculturing, and start to express dystrophin after fusion. These results demonstrate that the endometrial progenitor cells and menstrual blood–derived cells can transfer dystrophin into dystrophied myocytes through cell fusion and transdifferentiation in vitro and in vivo.

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Faculty Opinions recommendation of Toll-like receptor 4 ablation in mdx mice reveals innate immunity as a therapeutic target in Duchenne muscular dystrophy.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725296116.793509475 ◽

2015 ◽

Author(s):

James Tidball

Keyword(s):

Innate Immunity ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Therapeutic Target ◽

Mdx Mice ◽

Toll Like Receptor ◽

Toll Like Receptor 4

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Proliferative Effect of Tilapia Fish (Oreochromis niloticus) Lectin on BALB/c Mice Splenocytes

Protein and Peptide Letters ◽

10.2174/0929866526666190911144057 ◽

2019 ◽

Vol 26 (12) ◽

pp. 887-892

Author(s):

Cynarha Daysy Cardoso da Silva ◽

Cristiane Moutinho Lagos de Melo ◽

Elba Verônica Matoso Maciel Carvalho ◽

Mércia Andréa Lino da Silva ◽

Rosiely Félix Bezerra ◽

...

Keyword(s):

Cell Proliferation ◽

Oreochromis Niloticus ◽

Cytokine Production ◽

Thymidine Incorporation ◽

Con A ◽

Cell Viability Assay ◽

Proliferative Effect ◽

Viability Assay ◽

Apoptosis And Necrosis

Background: Lectins have been studied in recent years due to their immunomodulatory activities. Objective: We purified a lectin named OniL from tilapia fish (Oreochromis niloticus) and here we analyzed the cell proliferation and cytokine production in Balb/c mice splenocytes. Methods: Cells were stimulated in vitro in 24, 48, 72 hours and 6 days with different concentrations of OniL and Con A. Evaluation of cell proliferation was performed through [3H]-thymidine incorporation, cytokines were investigated using ELISA assay and cell viability assay was performed by investigation of damage through signals of apoptosis and necrosis. Results: OniL did not promote significant cell death, induced high mitogenic activity in relation to control and Con A and stimulated the cells to release high IL-2 and IL-6 cytokines. Conclusion: These findings suggest that, like Con A, OniL lectin can be used as a mitogenic agent in immunostimulatory assays.

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

Biomedicines ◽

10.3390/biomedicines9050481 ◽

2021 ◽

Vol 9 (5) ◽

pp. 481

Author(s):

Paulina Podkalicka ◽

Olga Mucha ◽

Katarzyna Kaziród ◽

Iwona Bronisz-Budzyńska ◽

Sophie Ostrowska-Paton ◽

...

Keyword(s):

Blood Flow ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Blood Vessels ◽

Mdx Mice ◽

Hindlimb Ischemia ◽

Double Positive ◽

Functional Studies ◽

Age Dependent ◽

Dystrophic Mice

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.

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Have Duchenne Muscular Dystrophy Patients an Increased Cancer Risk?

Journal of Neuromuscular Diseases ◽

10.3233/jnd-210676 ◽

2021 ◽

pp. 1-5

Author(s):

Gian Luca Vita ◽

Luisa Politano ◽

Angela Berardinelli ◽

Giuseppe Vita

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Systematic Investigation ◽

Mdx Mice ◽

True Incidence ◽

Patients With Cancer ◽

Dmd Gene ◽

Age Range ◽

Prevalence Of Cancer

Background: Increasing evidence suggests that Duchenne muscular dystrophy (DMD) gene is involved in the occurrence of different types of cancer. Moreover, development of sarcomas was reported in mdx mice, the murine model of DMD, in older age. So far, nine isolated DMD patients were reported with concomitant cancer, four of whom with rhabdomyosarcoma (RMS), but no systematic investigation was performed about the true incidence of cancer in DMD. Methods: All members of the Italian Association of Myology were asked about the occurrence of cancer in their DMD patients in the last 30 years. Results: Four DMD patients with cancer were reported after checking 2455 medical records. One developed brain tumour at the age of 35 years. Two patients had alveolar RMS at 14 and 17 years of age. The fourth patient had a benign enchondroma when 11-year-old. Conclusion: Prevalence of cancer in general in the Italian DMD patients does not seem to be different from that in the general population with the same age range. Although the small numbers herein presented do not allow definitive conclusion, the frequent occurrence of RMS in DMD patients raises an alert for basic researchers and clinicians. The role of DMD gene in cancer merits further investigations.

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Autophagy in the heart is enhanced and independent of disease progression in mus musculus dystrophinopathy models

JRSM Cardiovascular Disease ◽

10.1177/2048004019879581 ◽

2019 ◽

Vol 8 ◽

pp. 204800401987958

Author(s):

HR Spaulding ◽

C Ballmann ◽

JC Quindry ◽

MB Hudson ◽

JT Selsby

Keyword(s):

Skeletal Muscle ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Disease Progression ◽

Gene Mutations ◽

Dystrophin Gene ◽

Mdx Mice ◽

Dystrophin Deficiency ◽

Muscle Wasting Disease ◽

Dystrophin Protein

Background Duchenne muscular dystrophy is a muscle wasting disease caused by dystrophin gene mutations resulting in dysfunctional dystrophin protein. Autophagy, a proteolytic process, is impaired in dystrophic skeletal muscle though little is known about the effect of dystrophin deficiency on autophagy in cardiac muscle. We hypothesized that with disease progression autophagy would become increasingly dysfunctional based upon indirect autophagic markers. Methods Markers of autophagy were measured by western blot in 7-week-old and 17-month-old control (C57) and dystrophic (mdx) hearts. Results Counter to our hypothesis, markers of autophagy were similar between groups. Given these surprising results, two independent experiments were conducted using 14-month-old mdx mice or 10-month-old mdx/Utrn± mice, a more severe model of Duchenne muscular dystrophy. Data from these animals suggest increased autophagosome degradation. Conclusion Together these data suggest that autophagy is not impaired in the dystrophic myocardium as it is in dystrophic skeletal muscle and that disease progression and related injury is independent of autophagic dysfunction.

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BDMC protects AD in vitro via AMPK and SIRT1

Translational Neuroscience ◽

10.1515/tnsci-2020-0140 ◽

2020 ◽

Vol 11 (1) ◽

pp. 319-327

Author(s):

Chenlin Xu ◽

Zijian Xiao ◽

Heng Wu ◽

Guijuan Zhou ◽

Duanqun He ◽

...

Keyword(s):

Neurodegenerative Disorder ◽

Neuroprotective Effect ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

Activity Measurement ◽

Therapeutic Approaches ◽

Cell Viability Assay ◽

Viability Assay ◽

Compound C

AbstractBackgroundAlzheimer’s disease (AD) is a common neurodegenerative disorder without any satisfactory therapeutic approaches. AD is mainly characterized by the deposition of β-amyloid protein (Aβ) and extensive neuronal cell death. Curcumin, with anti-oxidative stress (OS) and cell apoptosis properties, plays essential roles in AD. However, whether bisdemethoxycurcumin (BDMC), a derivative of curcumin, can exert a neuroprotective effect in AD remains to be elucidated.MethodsIn this study, SK-N-SH cells were used to establish an in vitro model to investigate the effects of BDMC on the Aβ1–42-induced neurotoxicity. SK-N-SH cells were pretreated with BDMC and with or without compound C and EX527 for 30 min after co-incubation with rotenone for 24 h. Subsequently, western blotting, cell viability assay and SOD and GSH activity measurement were performed.ResultsBDMC increased the cell survival, anti-OS ability, AMPK phosphorylation levels and SIRT1 in SK-N-SH cells treated with Aβ1–42. However, after treatment with compound C, an AMPK inhibitor, and EX527, an SIRT1inhibitor, the neuroprotective roles of BDMC on SK-N-SH cells treated with Aβ1–42 were inhibited.ConclusionThese results suggest that BDMC exerts a neuroprotective role on SK-N-SH cells in vitro via AMPK/SIRT1 signaling, laying the foundation for the application of BDMC in the treatment of neurodegenerative diseases related to AMPK/SIRT1 signaling.

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