TISSUE-SPECIFIC STEM CELLS Nitric Oxide Controls Fat Deposition in Dystrophic Skeletal Muscle by Regulating Fibro-Adipogenic Precursor Differentiation NICOLETTA CORDANI,a VIVIANA PISA,b LAURA POZZI,a CLARA SCIORATI,c EMILIO CLEMENTIa,b Key Words. Skeletal muscle dystrophy • Fibro-adipogenic precursors • Nitric oxide • Fibrosis Adipocytes • MicroRNA
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Scientific Institute, IRCCS E. Medea 23842 Bosisio Parini, Lecco, Italy; bUnit of Clinical Pharmacology, Department of Biomedical and Clinical Sciences L. Sacco, University Hospital “Luigi Sacco,” Universita di Milano, Milan, Italy; cDivision of Regenerative Medicine, Ospedale San Raffaele Scientific Institute, Milan, Italy Correspondence: Emilio Clementi, M.D., Ph.D., Unit of Clinical Pharmacology, Department of Biomedical and Clinical Sciences, University Hospital “Luigi Sacco,” University of Milano, 20157 Milan, Italy. Telephone: 39-0250319640; Fax: 39-02-50319646; e-mail: emilio.clementi@ unimi.it; or Clara Sciorati, Ph.D, Division of Regenerative Medicine Ospedale San Raffaele Scientific Institute, via Olgettina 58, 20132 Milan, Italy. Telephone: 39-02-26436342; Fax: 39-02-26435283; e-mail: [email protected] Received July 18, 2013; accepted for publication September 5, 2013; first published online in STEM CELLS EXPRESS October 29, 2013. C AlphaMed Press V
1066-5099/2014/$30.00/0 http://dx.doi.org/ 10.1002/stem.1587
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ABSTRACT Duchenne muscular dystrophy (DMD) is an hereditary disease characterized by loss of muscle fibers and their progressive substitution by fat and fibrous tissue. Mesenchymal fibro-adipogenic progenitors (FAPs) expressing the platelet-derived growth factor receptor alpha (PDGFRa) are an important source of fibrosis and adipogenesis in dystrophic skeletal muscle. Among the therapies suggested for dystrophy are those based on nitric oxide (NO) donating drugs, the administration of which slows disease progression. NO has been shown to act by enhancing the regenerative potential of the diseased muscle. Whether it acts also by inhibiting fibrosis and adipogenesis was not known. Here, we show in vitro that NO regulates FAP fate through inhibition of their differentiation into adipocytes. In mdx mice, an animal model of DMD, treatment with the NO donating drug molsidomine reduced the number of PDGFRa1 cells as well as the deposition of both skeletal muscle fat and connective tissues. Inhibition of adipogenesis was due to NO-induced increased expression of miR-27b leading to downregulation of peroxisome proliferator-activated receptors gamma (Pparc1) expression in a pathway independent of cGMP generation. These findings reveal an additional effect of NO in dystrophic muscle that conceivably synergizes with its known effects on regeneration improvement and explain why NO-based therapies appear effective in the treatment of muscular dystrophy. STEM CELLS 2014;32:874–885
INTRODUCTION Duchenne muscular dystrophy (DMD) is a rare disease caused by mutations in the dystrophin encoding-gene, leading to protein loss with an incidence of one out of 3,600–6,000 newborn males [1]. Dystrophin mutations result in skeletal muscle fiber damage and necrosis, leading to progressive substitution of fibers with connective and adipose tissue that leads to death in early adulthood [1]. The pathophysiology of muscular dystrophies also correlates with an altered synthesis of nitric oxide (NO) that is a key signaling molecule controlling adult skeletal muscle structure, bioenergetics, and excitation-contraction coupling [2–5]. In DMD patients, as well as in mouse models of muscular dystrophies such as the mdx mouse, the splice variant of neuronal NO synthase (nNOS) is displaced from the sarcolemma and relocates to the cytosol, an event accompanied by a substantial reduction of enzymatic activity [6–10]. Restoration of NO generation by transgenic expression of nNOS in the mdx mouse model ameliorates its dystrophic phenotype [11, 12] and treatment with
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NO donating drugs reduces muscle necrosis as well as connective tissue deposition [13–16]. We have demonstrated that NO donating drugs administered together with nonsteroidal anti-inflammatory drugs have long-term beneficial effect in mouse models of dystrophy with recovery of skeletal muscle morphology and function [17–19]. The potential of this treatment was explored in a pilot study in dystrophic patients where it showed a high profile of safety and tolerability with promising signs of efficacy [20]. Several groups have explored the mechanisms beyond the therapeutic potential of NO and found that it has multiple actions on survival, activation and differentiation of satellite cells, the mononuclear myogenic precursor cells. These actions enhance the ability of these cells to repair the damaged muscle [21– 26]. Recently, using the NO-donating drug molsidomine, we found that the beneficial effect of NO is explained in part by the ability of the drug to enhance self-renewal of satellite cells, thus delaying the exhaustion of the satellite cells pool and maintaining their regenerative potential [27]. C AlphaMed Press 2013 V
Cordani, Pisa, Pozzi et al. Progressive reduction of muscle regeneration capacity in DMD muscle due to satellite cells exhaustion in DMD correlates with the substitution of degenerated fibers with adipocytes and fibrous tissue. To this substitution contributes significantly a population of mesenchymal fibro-adipogenic progenitors (FAPs) residing in the muscle, specifically located in the interstitial spaces [28–31]. These cells can differentiate efficiently into adipocytes [28, 29], fibroblasts as well as osteoblastic and smooth muscle cells under specific culture conditions [28–30]. While not generating myofibers, they proliferate following muscle damage, fulfilling a trophic support function for myoblasts, to eventually return to a quiescent state in healed muscles [31]. When regeneration fails, however, FAPs generate the components of the fibro-fatty tissue infiltrates that are characteristic of the degenerating muscle [31–33]. Here, we show that long-term treatment of mdx mice in vivo with molsidomine reduces FAP numbers in skeletal muscle tissue and inhibits adipose tissue deposition and indirectly fibrosis. These are direct effects of NO due to a NOdependent regulation of both miR-133a, a known regulator of Collagen 1A1 expression [34] and miR-27b, a key inhibitor of adipocytes differentiation controlling the expression of peroxisome proliferator-activated receptor c (Pparc) [35–37]. In particular, the effect of NO in vitro on adipogenesis was due to cGMP-independent inhibition of Pparc via control of promoter activity and enhancement of expression of miR-27b. These results indicate an action of NO donating drugs additional to the already known effects on muscle myofibers and satellite cells that is also involved in muscle repair. The fact that NO acts by preventing multiple mechanisms of muscle degeneration contributes to explain why NO-donating drugs are effective in dystrophy and suggests that their therapeutic potential should be explored further. In this respect, it is important to note that molsidomine has a good profile of safety and tolerability in humans [38].
MATERIALS
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Animals and Treatments C57BL/6J wild-type (WT) mice (Charles River, Wilmington, MA; http://www.criver.com) and mdx24cv mice (B6Ros.CgDmdmdx-4cv/J, C57/BL/6 background) (Jackson Laboratory, Bar Harbor, ME; http://www.jax.org) were treated in accordance with European Community guidelines and with the approval of the Institutional Ethical Committee. Mice were fed either with standard diet or a diet containing 3 mg/kg of molsidomine (1-ethoxy-N-(3-morpholino-5-oxadiazol-3-iumyl) methanimidate, Mucedola S.r.l., Milan, Italy), according to the measured daily food intake. Mdx mice (eight animals per group) were treated starting at 1 month of age and sacrificed 5 months upon treatment for histological and molecular analyses.
Isolation of FAPs For FAP cells isolation, hind limb muscles of 8–10 weeks old mice were minced and digested with 0.2% of Collagenase II (Worthington Biochemical, Lakewood, NJ, http://worthingtonbiochem.com) following published protocols [29]. Cells were stained with the following antibodies: anti-CD31phycoerythrin (PE)/Cy7 (PECy7, clone 390), anti-CD45-PECy7
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(clone 30-F11) (eBioscience, Inc., San Diego, CA, http://www. ebioscience.com), anti-SM/C2.6-Biotin (kindly provided by Dr. Fukada) [39], streptavidin-phycoerythrin (PE, BioLegend, San Diego, CA, http://www.biolegend.com), anti-a7-Integrin-PE (AbLab, UBC Antibody Lab, Vancouver, Canada, http://www. ablab.ca), anti-platelet-derived growth factor receptor alpha (PDGFRa)-allophycocyanin (APC, CD140a clone APA5, BioLegend), anti-LY-6A/E SCA-1-allophycocyanin/Cy7 (APC/Cy7, clone B7, BD Biosciences, Franklin Lakes, NJ, http://www.bdbiosciences.com), and 7-aminoactinomycin D (Life Technologies, Paisley, U.K., http://www.lifetech.com). FAP cells were selected as CD452/ CD312/SM/C2.62 or a7-Integrin2/SCA-11 and PDGFRa1 cells.
Cell Culture Freshly isolated FAP cells were plated at 1 3 104 cells per centimeter square in multiwell plates or in chamber slides and maintained for 6 days in growth medium (GM) consisting of Dulbecco’s modified high glucose Eagle’s medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin plus 5 ng/ml of recombinant human basic fibroblast growth factor (PeproTech, Rocky Hill, NJ; http://www.peprotech.com) in a humidified incubator (37 C and 5% CO2) using plates coated with growth factor reduced BD Matrigel Matrix (BD Biosciences 2350 Qume Drive San Jose, CA http://www.bdbiosciences.com). For differentiation into fibroblasts, FAP cells were cultured for 5 days in DMEM containing 2% FBS and 2 ng/ml of transforming growth factor b 1 (TGFb-1) (Sigma-Aldrich, St. Louis, MO; http://www.sigmaaldrich.com) on eight-well LabTeck Chamber slides (Nunc, Thermo Fisher Scientific Inc Waltham, MA; https://http://www.nuncbrand.com). For adipogenic differentiation, FAP cells were cultured in an adipogenic medium (AM) consisting of DMEM—Ham’s F-12 Nutrient Mixture (DMEM-F-12) with 10% FBS and supplemented with 0.5 mM the phosphodiesterase inhibitor 3isobutyl-1-methylxanthine, 1 mM dexamethasone, and 1 mg/ml insulin (all from Sigma Aldrich). After 3 days in AM, cells were switched to DMEM-F-12 with 10% FBS and 10 mg/ml insulin, which was changed every 2 days. Experiments were carried out using 50 mM of the NO donor 1-[N-(2-aminoethyl)-N-(2ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO, Calbiochem, Merck-Millipore, Frankfurt, Germany; http://www. merckmillipore.com), except for the experiments of Supporting Information Figure S2 where the concentration used were also 10 mM and 20 mM, 10 mM of the guanylate cyclase inhibitor 1H-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, Alexis Biochemicals, Vinci-Biochem S.r.l., FI; http://www.enzolifesciences. com) or vehicle.
Gene and MicroRNA Expression Analysis Total RNA was isolated from cultured cells and homogenized tibialis anterior (TA) muscles with Qiagen RNeasy Micro kit (Qiagen, Hamburg, Germany; http://www.qiagen.com) or the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions, and then reverse-transcribed into cDNA using SuperScript VILO cDNA synthesis kit (Invitrogen- Life Technology, Carlsband, CA http://www.invitrogen.com). Specific sets of primer pairs, as described in Supporting Information Figure 1, were designed to amplify the desired gene sequence. Realtime PCR (RT-PCR) was performed using the SYBR Green Supermix (Biorad, Hercules CA) on a Roche LightCycler 480 C AlphaMed Press 2013 V
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Figure 1. Nitric oxide (NO) downregulates the number of PDGFRa1 cells. (A): Number of PDGFRa1 in tibialis anterior muscle of WT or in mdx mice treated with standard diet (MDX SD) or a diet containing the NO donating drug molsidomine (MDX MOLS): representative images of PDGFRa immuno-staining are also shown (right: 320, scale bars 5 50 mm.). (B): Pdgfra mRNA expression. Data are shown as mean 6 SE; n 4 **, p < .01; ***, p < .001 versus MDX SD. Abbreviations: MOLS, molsidomine or 1-ethoxy-N-(3-morpholino-5-oxadiazol-3-iumyl)methanimidate; PDGFRa, platelet-derived growth factor receptor alpha; WT, wild type.
Instrument (Roche Diagnostics, Rotkreuz, Switzerland; https:// http://www.roche-applied-science.com). All reactions were run in duplicates. Expression levels were analyzed using the 2(2DDCt) method and relative expression levels (arbitrary units, A.U.) using the 2(2DCt) method [40]. Gapdh (for cells) or Rpl32 (for tissues) were used as reference genes to normalize mRNA expression levels. Each plot shown in figures is a mean of at least three independent experiments. RT-PCR-based detection of mature microRNAs (miRNAs) (miR-133a, miR-27b, miR-27a, miR-130a, and miR-181a) and mouse-specific reference snoRNAs (SNO 55 and SNO U6) was carried out from FAPs and tissue samples isolated using the microRNA Reverse Transcription Kit and gene-specific primers and probes (TaqMan MicroRNA Assays), according to the manufacturer’s specifications. All reactions were run in duplicates; snoRNA 55 and snoRNA U6 were used as endogenous genes in tissue and cells analyses, respectively. For these experiments, miRNA expression levels were calculated using the 2(2DCt) formula. All reagents, including primers and kits, were obtained from Applied Biosystems (Life Technologies, Carlsbad, CA). C AlphaMed Press 2013 V
Immunofluorescence Animals were killed by cervical dislocation, TA muscles dissected and immediately frozen in liquid N2-cooled isopentane. Analyses were performed on 10 mm serial sections fixed for 10 minutes in 4% paraformaldehyde. Sections were then blocked 30 minutes with 10% goat serum, 0.1% Triton X-100, incubated overnight with primary antibodies at 4 C followed by appropriate secondary antibodies (Alexa Fluor 488 or 546conjugated; Invitrogen). The following antibodies were used: anti-mouse PDGFRa (R&D System, Minneapolis MN; http:// www.rndsystems.com), anti-mouse PERILIPIN A (Sigma), antimouse LAMININ (Abcam, Cambridge, U.K.; http://www.abcam. com), anti-peroxisome proliferator-activated receptors gamma (PPARc) (E-8, Santa Cruz, CA; http://www.scbt.com), and antimouse COL3A1 (S-17, Santa Cruz). Nuclei were counterstained with Hoechst 33342 (Molecular Probes, Life Technologies) and slides were finally mounted with the ProLong Gold antifade reagent (Invitrogen, Life Technologies). Stained tissues were photographed with a LEICA CTR4000 fluorescence microscope STEM CELLS
Cordani, Pisa, Pozzi et al. LEICA, Allendale NJ, and processed using the ImageJ software (http://rsb.info.nih.gov/ij). FAP cells number was calculated by counting the number of interstitial PDGFRa1 cells per field while fat deposition was measured by calculating PERILIPIN area (mm2) per field in four representative images of four independent animals per group. Immunofluorescence on FAP cells was performed following the protocol described above.
Oil Red O Staining FAP cells were fixed in 4% paraformaldehyde for 10 minutes and then stained for 1 hour by incubating with 0.5% oil red O (Sigma) in a 60:40 isopropanol/water mixture, filtered, and washed twice in distilled water. Nuclei were counterstained with Mayer Hematoxylin and then photographed with a LEICA CTR4000 microscope.
Western Blot Analyses Total lysates were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, and probed using the following antibodies: anti-mouse C-EBPb (C-19, Santa Cruz), anti-mouse Kruppel-like factor 4 (KLF4) (Genespin, Milan, Italy), anti-mouse GADD153/ CHOP (B-3, Santa Cruz), anti-mouse PPARc (H-100x Santa Cruz), anti-mouse C-EBPa (14AA, Santa Cruz), anti-mouse PERILIPIN A (Sigma), and anti-mouse VINCULIN (Sigma). Chemiluminescent HRP-conjugated secondary antibodies were developed using the Super Signal West Femto substrate (Pierce, Thermo Fisher Scientific, Inc., Whaltham, MS) or the Clarity substrate (Biorad) and acquired with a ChemiDoc XRS imager (Bio-Rad). Analyses were performed using the Image J and Image Lab softwares (Biorad). VINCULIN was used to normalize protein expression level.
Luciferase Reporter Assay For reporter analyses, FAP cells were seeded as previously described in 24-well plates. When they reached 80% confluence, cells were transfected with Lipofectamine 2000 (Invitrogen, Life Technologies) following the manufacturer’s instructions. Six hours after transfection, adipogenic differentiation was initiated by AM supplemented with DETA-NO or vehicle. Pparc1 promoter activity was assessed using the pGL3PPARc1 (pGL3-PPARc1 p3000) construct, a gift by Dr. Brune, who kindly provided also and pGL3-PPARc-30 -UTR and pGL3PPARc-30 -UTR-C83A/U84G constructs [35]. The vector pGL3basic was kindly provided by D. Gabellini (Ospedale San Raffele). The latter two constructs were used to test PPARc-30 UTR, where the miR-27 binding site is located. Cells were harvested 3 days after transfection, kept in AM in presence of DETA-NO or vehicle. Firefly luciferase and Renilla activities were measured using the Dual Luciferase Reporter Assay System and the Glo-Max Multi detection System (Promega, Madison, MS; http://www.promega.com), respectively.
Transfections FAP cells were seeded as described above, in six-well plates and transfected when they reached 80% confluence either with 50 nM anti-miR-27b (hsa-miR-27b-3p inhibitor— MIMAT0000126—Ambion, Life Technologies) or 50 nM antimiR inhibitor as a negative control (mirVana miRNA Inhibitor, Negative Control #1, Ambion, Life Technologies), using RNAIMAX (Life Technologies) as per manufacturer’s instructions. Six hours after transfection adipogenic differentiation was started
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by changing medium to AM supplemented either with DETANO or vehicle as a control. Transfected cells were harvested and analyzed 6 days later.
Statistical Analyses All values are expressed as mean 6 SEM of at least three independent experiments. All data were analyzed using either an unpaired two-tailed Student’s t test for comparisons between two groups, and one-way ANOVA with appropriate post-test adjustment for multiple group comparisons. p-Values lower than 5% (p < .05) were considered statistically significant, single, double, triple, and quadruple asterisks in the figures indicate p < .05, p < .01, p < .001, and p < .0001, respectively.
RESULTS NO Decreases the Number of PDGFRa1 Cells in Dystrophic Muscle We evaluated the effect on FAP number in dystrophic muscle of oral administration of molsidomine for 5 months to mdx mice. PDGFRa1 cells were barely detectable in TA of WT animals and abundant in mdx mice treated with standard diet. Molsidomine treatment significantly decreased the number of PDGFRa1 cells (Fig. 1A). RT-PCR analyses confirmed these data by showing a 3.5-fold decrease of Pdgfra expression in mdx mice treated with molsidomine compared to those treated with the standard diet (Fig. 1B).
Collagen 1A1 and 3A1 Are Reduced in NO-Treated Mdx Muscle PDGFRa1 cells are abundant in fibrotic muscle and express fibrosis-related markers [30]. We investigated the expression of Collagen 1A1 (Col1A1) and Collagen 3A1 (Col3A1), two markers of fibrosis, in TA muscles. Mdx mice fed with standard diet expressed high levels of both collagen isoforms in comparison to WT mice, while the 5 months treatments with molsidomine treatment reduced significantly the levels of both mRNAs (Fig. 2A). Consistently with these data, mdx mice showed reduced levels of miR-133a, a known negative regulator of Col1A1 [34], which was increased by molsidomine treatment (Fig. 2B).
NO Reduces Fat Deposition in Dystrophic Mice We evaluated fat deposition in mdx mice by assessing the levels of PERILIPIN, a protein coating lipid droplets of adipocytes [41]. As reported in Figure 3A, PERILIPIN positivity was significantly increased in mdx mice with respect to WT mice and normalized after treatment with molsidomine. To evaluate better the inhibitory effect of NO on fat deposition, we measured the expression of two biomarkers of mature adipocytes, the fatty acid binding protein 4 (Fabp4) and Adiponectin, and of two adipose-regulating transcription factors, Pparc1 and the CCAAT/enhancer-binding protein a (C-Ebpa). Pparc1, C-Ebpa, Fabp4, and Adiponectin mRNA expression levels in TA of mice treated for 5 months with molsidomine were almost halved when compared with mice that received standard diet, and comparable to those of WT mice (Fig. 3B). Consistently with these data, the expression of miR-27b a negative regulator of Pparc that is abundantly expressed in WT mice was significantly reduced in standard diet-receiving mice and reinduced in molsidomine-treated animals (Fig. 3C). C AlphaMed Press 2013 V
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Figure 2. Nitric oxide (NO) inhibits collagen deposition. (A): Collagen 1A1/3A1 mRNA expression and (B) miR-133a expression in muscle of WT or mdx mice treated with standard diet (MDX SD) or a diet containing the NO donating drug molsidomine (MDX MOLS). Data are shown as mean 6 SE; n 5. *, p < .05; ***, p < .001 versus MDX SD. Abbreviations: MOLS, molsidomine or 1-ethoxy-N-(3-morpholino-5oxadiazol-3-iumyl)methanimidate; WT, wild type.
NO Inhibits Differentiation of FAPs into the Adipogenic Lineage While Not Affecting Their Differentiation into Collagen-Producing Cells We investigated whether inhibition of collagen and fat deposition induced by NO donation in vivo can be ascribed to regulation of FAP fate. We purified from the hind limb muscles of WT and mdx mice FAPs, that were defined as mesenchymal cells that are CD452 (hematopoietic lineage negative), CD312 (endothelial negative), SMC2.62 or a7 integrin2 (satellite cells negative), and SCA-11/PDGFRa1 cells [28, 29]. FAPs were subsequently cultured in a profibrotic medium containing TGFb1 [30], either in the presence of the NO donor DETA-NO or vehicle. Immunofluorescence with anti-COL3A1 antibody was carried out to evaluate the effects of NO on differentiation of matrix-producing cells. The percentage of collagen1 cells remained unchanged after DETA-NO treatment when compared with vehicle both in FAPs isolated from WT and mdx mice (Fig. 4A). To investigate the effect of NO donation on FAPs adipogenic potential, we performed oil red O dye staining on WTand mdx-derived FAPs cultured under adipogenic conditions using a specific induction medium (AM). DETA-NO strongly decreased the number of Red Oil1 cells both in WT and mdxderived FAPs cells with respect to vehicle suggesting that DETA-NO drastically affects FAPs adipogenic potential (Fig. 4B). The effect of DETA-NO was concentration dependent (Supporting Information Fig. S2).
NO Inhibits FAPs Adipogenic Potential in a cGMP-Independent Way To investigate the mechanism of NO-induced inhibition of FAPs differentiation into adipocytes, we first focused our attention on the expression of the transcription factors directC AlphaMed Press 2013 V
ing the adipogenic lineage. DETA-NO did not modify KLF4, CEBPb, and CHOP/GADD153 protein levels early after adipogenesis induction (Supporting Information Fig. S3), while it significantly inhibited PPARc, a transcription factor present in committed preadipocytes and mature cells [42], and reduced the expression of PERILIPIN (Fig. 5A). NO acts via generation of cyclic GMP (cGMP) and/or via cGMP-independent mechanisms. To assess the cGMP dependency of the NO action, we induced FAPs differentiation in AM in presence of DETA-NO, either alone or in combination with the guanylate-cyclase inhibitor ODQ. The inhibitory effect of DETA-NO on the mRNA expression levels of Pparc1, C-Ebpa, Fabp4, and Adiponectin was not reversed by ODQ, which per se did not have any effect (Fig. 5B). Likewise, ODQ did not modify the effect of DETA-NO on PPARc1, C-EBPa, fatty acid binding protein 4, and PERILIPIN protein levels (Fig. 5C).
NO Regulates PPARc Via MiR-27b We investigated how NO inhibits Pparc expression. To this end, we analyzed the TF promoter and its transcript regulation. We performed luciferase reporter assays in FAPs transiently transfected with a construct containing the luciferase encoding region under the control of the Pparc promoter 1(PGL3-c1) [35]. After transfection, FAPs cells were cultured in AM containing DETA-NO or vehicle. AM induced a significant increase in Pparc promoter 1 versus GM (Supporting Information Fig. S4). A 40% reduction of activity was detected in the presence of DETA-NO (Fig. 6A). Given the greater reduction of Pparc1 mRNA levels induced by DETA-NO in comparison with Pparc promoter activity downregulation (Fig. 5B vs. Fig. 6A), we tested the hypothesis that an additional posttranscriptional regulation of Pparc1 by NO was present. We measured the activity of both WT PPARc-30 -UTR and mutated STEM CELLS
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Figure 3. Nitric oxide (NO) inhibits fat deposition. (A): PERILIPIN staining in WT or mdx mice treated with standard diet (MDX SD) or a diet containing the NO donating drug molsidomine (MDX MOLS). Upper panel shows data quantification as PERILIPIN1 area (mean6 SE; n 5 7); lower panels show representative images of PERILIPIN immuno-staining (320, scale bars 5 50 mm). (B): Pparc1, C-ebpa, Fabp4, and Adiponectin mRNA and (C) miR-27b expression (mean 6 SE, n 3, *, p < .05; **, p < .01; ***, p < .001 vs. MDX SD). Abbreviations: MOLS, molsidomine or 1-ethoxy-N-(3-morpholino-5-oxadiazol-3-iumyl)methanimidate; WT, wild type.
30 -UTR containing vectors normalized versus pGL3-basic in transiently transfected FAPs cultured in AM. DETA-NO reduced significantly the luciferase activity of the PPARc-30 -UTR but was ineffective when miR-27 binding site was mutated (Fig.
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6B). When we examined the expression of miRNAs possibly implicated in adipogenic differentiation, namely miR-27a/ 130a/181a and miR-27b, we found that only miR-27b was affected by DETA-NO treatment (Fig. 6C and Supporting C AlphaMed Press 2013 V
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Figure 4. NO modulates in vitro fibro-adipogenic progenitors (FAPs) adipogenic potential but not FAP differentiation into collagenexpressing cells. (A): Data quantification (left) and representative images (320, scale bars 5 50 mm; right) of COLLAGEN 3A1 staining in TGFb-treated FAPs isolated from WT or mdx mice in the presence of the NO donor DETA-NO or vehicle. (B): Oil red O-staining in FAPs isolated and treated as described above and cultured in adipogenic conditions. Data quantification is in the upper panel while representative images of the staining (310, scale bars 5 100 mm or 340 magnification of the boxes, scale bars 5 25 mm) in the lower panels. Data are expressed as mean 6 SE; n 5, ***, p < .001; ****, p < .0001 versus vehicle. Abbreviations: DETA-NO, DETA-NONOate (1-[N-(2aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate); WT, wild type.
Information Fig. S5). In particular, miR-27b expression levels were high in proliferating FAPs (GM) and reduced by almost threefold when adipogenesis was induced by switch to AM. DETA-NO increased twofold the expression of miR-27b when compared with vehicle. To confirm these data further, we evaluated the effect of DETA-NO treatment on Pparc1 mRNA expression level after transient transfection with antagomiR27b. As shown in Figure 6D, miR-27b was silenced by its own C AlphaMed Press 2013 V
antagomiR with respect to the negative control (a-miR-27b vs. a-miR neg, upper panel). NO-induced Pparc1 inhibition completely disappeared when antagomiR-27b was present.
DISCUSSION Adult skeletal muscle regenerates upon damage thanks to the ability of resident proliferating satellite cells capable of forming STEM CELLS
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Figure 5. NO inhibits fibro-adipogenic progenitors (FAPs) adipogenic differentiation by a cGMP-independent mechanism. (A): Representative images of PPARc and PERILIPIN immuno-staining in FAPs treated with DETA-NO or vehicle (320, scale bars 5 50 mm). (B): Expression of the mRNA of adipogenesis-markers (Pparc1, C-Ebpa, Fabp4, and Adiponectin) in FAPs treated with DETA-NO, the guanylatecyclase inhibitor ODQ, or vehicle. (C): Representative images (upper panels) and quantification (lower panels) of adipogenesis-markers (PPARc1, C-EBPa, and PERILIPIN) protein expression in FAPs treated as above. VINCULIN was used as an internal control. Data are presented as fold change to vehicle-treated cells (fixed at 1) and reported as mean 6 SE, n 4. *, p < .05; **, p < .01; ***, p < .001 versus vehicle. Abbreviations: DETA-NO, DETA-NONOate (1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate); PPARc, peroxisome proliferator-activated receptors gamma.
new fibers. When chronic damage occurs, such as in dystrophy, and compensative regeneration fails because of progressive exhaustion of the satellite cells pool [43–45], fibro-adipogenic degeneration becomes predominant, in a process termed “reparative disorder” [33]. An important role in this process has been recently demonstrated for a group of mesenchymal stem cells, FAPs, capable of differentiating in vitro toward both fibrogenic and adipogenic lineages [31]. FAPs are located into the interstitial space between myofibers close to vessels and can be purified as CD452, CD312, a7-Integrin2, CD341, Sca11, and PDGFRa1 cells [28, 29]. Upon damage FAPs expand,
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prior to satellite cells proliferation, and invade the surrounding tissue. Subsequently, FAPs are either cleansed from the invaded tissue (in case of a successful repair process) or, if the regeneration process is defective or continuous as in degenerative muscle disorders, they differentiate into adipocytes [46]. Investigation of the cellular and molecular effectors of the regeneration/degeneration switch is relatively recent and pathways and regulatory mechanisms still need to be identified in full [47, 48]. The role of these cells and their regulation appears to be important in the overall process of muscle healing alongside the much better characterized pathogenesis of skeletal muscle C AlphaMed Press 2013 V
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Figure 6. NO regulates Pparc promoter and affects its mRNA stability controlling miR-27b. (A): Luciferase reporter assay in fibroadipogenic progenitors (FAPs) transfected with the Pparc1 promoter construct or (B) with constructs of normal (pGL3-PPARc-30 -UTR) or mutated (pGL3-PPARc-30 -UTR-C83A/U84) 30 -UTR of Pparc promoter region and treated with DETA-NO or vehicle. Data are expressed as mean 6 SE after normalization to pGL3 basal activity. (C): Mir-27b expression in FAPs cultured in proliferating (GM) or in adipogenic conditions (AM) and treated with DETA-NO or vehicle. (D): MiR-27b (upper) or Pparc1 (lower) expression in FAPs cultured in adipogenic conditions in the presence or absence of DETA-NO and transfected with anti-miR-27b (a-miR-27b) or negative control (a-miR neg). Data are presented as mean of relative expression 6 SE, n 3. *, p < .05; **, p < .01 versus vehicle or versus DETA-NO 1 a-miR neg. for panel (D). Abbreviations: DETA-NO, DETA-NONOate (1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate); PPARc, peroxisome proliferator-activated receptors gamma.
disease and interesting novel therapeutic targets. The shedding of light on FAPs, especially considering the complexity of skeletal muscle cells network interactions, is thus crucial as it would add novel therapeutic options to muscular dystrophy. Here, we show that oral administration of the NO donating drug molsidomine in mdx mice decreases the number of FAPs in muscle while reducing collagen expression as well as adipose tissue deposition. We analyzed the mechanism of NO action. NO delivered with the NO donor DETA-NO inhibited the expression of the C AlphaMed Press 2013 V
adipocyte markers PERILIPIN, Adiponectin, and Fabp4 as well as adipogenic differentiation by both mdx and WT-derived FAPs. Interestingly, the action of NO was not exerted at the initial stages of adipogenic differentiation, as it affected expression only of the middle adipogenic transcription factors Pparc and C-ebpa that coordinate and direct the development of adipogenic lineage [42]. NO did not affect, in the experimental condition used, expression of the early adipogenesis transcription factors KLF4, c-EBPb, and CHOP10 while regulating PPARc1, a key transcription factor in adipogenesis [42, 49, 50]. PPARc is a STEM CELLS
Cordani, Pisa, Pozzi et al. member of the nuclear hormone receptors subfamily, highly enriched in adipose tissue. PPARc ectopic expression and activation in fibroblasts promote their conversion into lipid-filled adipocytes inducing a complete adipogenic gene expression program [51]. In particular, we focused on Pparc1 since it is able to drive in itself development of adipose tissue in mouse independently of Pparc2 [52]. We unequivocally demonstrate that NO regulates Pparc1 expression by regulating the expression of miR-27b, a specific adipogenesis-regulator that in turn controls Pparc mRNA stability [36, 37]. MicroRNAs (miRNAs) are small non-protein coding RNAs, some of which acting as post-transcriptional gene regulators in muscle development and function [53]; their expression is altered in several muscle disorders including DMD [54, 55]. No specific information about the function and regulation of miR-27b in DMD and in adipogenesis regulation in muscle however was until now available. Our results and in particular the observation that induction of miR-27b by NO occurred both in vitro (isolated FAPs) and in vivo (mdx mice) suggest that miR-27b may be the mechanism by which NO endogenously generated by myofibers reduces adipogenesis and fat tissue deposition in muscular dystrophy. We also found that NO enhances the expression of miR133a, the expression of which we found to be reduced in mdx muscles as already demonstrated [56]. We cannot at present identify a specific functional role of this inhibitory action by NO. The role of miR-133a, whose levels are lower in sera from DMD patients when compared with healthy individuals [56], is not yet clear as it seems to be dispensable for normal muscle development [57] although regulating Col1A1 synthesis in several tissues. We have not analyzed in detail how NO regulates miR-27b expression except for the cGMP-independency of its effect. Interestingly, alongside miR-133a, NO regulates also other miRNAs in skeletal muscle [56, 58, 59]. In particular, a single local injection in mdx muscle of the NO donating drug nitroglycerin increased the expression of miR-1 and miR-29. This was due to S-nitrosylation [60] and then removal of the inhibitory histone deacetylase 2 from the promoters of these miRNA [56]. Whether regulation of miRNAs 133a and 27b is mediated by the same mechanism remains to be investigated further. Interestingly, miR-1 is linked to the redox state of cell, and miR-29 is involved into the fibrotic process [56]. Our observation that NO also controls adipogenic differentiation via miR-27b indicates that this is a relevant mechanism by which NO controls muscle homeostasis. How this integrates with the other wellknown homeostatic actions of NO, for example, on muscle bioenergetics, glucose uptake, and excitation contraction coupling remains also to be investigated [5, 26, 61–63]. We found that molsidomine in vivo reduces collagen expression in mdx mice, while DETA-NO in vitro did not influence TGFb-induced FAPs differentiation into fibroblasts. This suggests the possibility that extracellular matrix producing cells other than FAPs are regulated by NO. Fibrocytes, and in particular myofibroblasts, are an important source of extracellular matrix deposition in dystrophic muscle. These cells are present during tissue repair and their activation persists because of constant myofibers breakdown and chronic inflammation [31, 64, 65].
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We have recently demonstrated using a-sarcoglycan null mice, another animal model of muscular dystrophy, that molsidomine is endowed with therapeutic effects, maintaining the functional pool of satellite cells and stimulating the regeneration capacity of skeletal muscle [27]. Moreover, molsidomine modulates the characteristics of the inflammatory cell infiltrate within a-sarcoglycan null dystrophic muscles, and this contributes to its healing function. In particular, molsidomine boosts the subset of macrophages associated to reduced fibrosis and increased preservation of the muscle tissue [66]. The combination of these different but synergic actions of molsidomine on muscle repair together with the good profile of safety and tolerability [20] are in keeping with the idea that molsidomine and other NO donating drugs are precious tools for DMD patients therapy that need to be investigated and refined further.
CONCLUSIONS This work demonstrates for the first time both in vivo and in vitro that NO regulates FAPs number and fate controlling fat tissue deposition in dystrophic skeletal muscle. The underlying mechanism of these actions is regulation of Pparc1 expression through direct modulation of the promoter activity and posttranscriptional control of miR-27b levels in a cGMPindependent way. Moreover, NO inhibits collagen deposition affecting FAPs in vivo. Altogether, these data point out the therapeutic role of NO donating drugs in the treatment of skeletal muscle dystrophy in virtue of their ability to affect multiple mediators playing a role in the complex pathogenic process of the disease.
ACKNOWLEDGMENTS We thank Prof. Bernard Br€ une (Goethe-University Frankfurt) for providing to us the pGL3-PPARc and related vectors. We are grateful to Prof. So-ichiro Fukada (Osaka University, Osaka, Japan) for the SM/C2.6 antibody. We thank Dr. Silvia Brunelli (University of Milano-Bicocca, Milan, Italy) for helpful discussion. This work was supported by the European Community’s framework programme FP7/2007–2013 under grant agreement n 241440 (ENDOSTEM) (to E.C.), the Italian Ministry of Health RC 2013 (to E.C.), Associazione Italiana Ricerca sul Cancro (AIRC IG11362) (to E.C.), and from the Ministero della Universita e Ricerca PRIN 2010–2011 (to E.C.).
AUTHORS CONTRIBUTIONS N.C.: conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing; V.P.: collection and assembly of data and data analysis and interpretation; L.P.: collection and assembly of data; C.S.: conception and design and manuscript writing; E.C.: conception and design, manuscript writing, financial support, and final approval of the manuscript. C.S. and E.C. are co-corresponding authors.
DISCLOSURE
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POTENTIAL CONFLICTS
OF INTEREST
The authors indicate no potential conflicts of interest.
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57 Deng Z, Chen JF, Wang DZ. Transgenic overexpression of miR-133a in skeletal muscle. BMC Musculoskelet Disord 2011;12:115. 58 Zhu H, Bhattacharyya BJ, Lin H et al. Skeletal muscle calpain acts through nitric oxide and neural miRNAs to regulate acetylcholine release in motor nerve terminals. J Neurosci 2013;33:7308–7324. 59 Cazzella V, Martone J, Pinnaro C et al. Exon 45 skipping through U1-snRNA antisense molecules recovers the Dys-nNOS pathway and muscle differentiation in human DMD myoblasts. Mol Ther 2012;20:2134– 2142. 60 Colussi C, Mozzetta C, Gurtner A et al. HDAC2 blockade by nitric oxide and histone deacetylase inhibitors reveals a common target in Duchenne muscular dystrophy treatment. Proc Natl Acad Sci USA 2008;105: 19183–19187. 61 Tengan CH, Rodrigues GS, Godinho RO. Nitric oxide in skeletal muscle: Role on mito-
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Scientific Institute, IRCCS E. Medea 23842 Bosisio Parini, Lecco, Italy; bUnit of Clinical Pharmacology, Department of Biomedical and Clinical Sciences L. Sacco, University Hospital “Luigi Sacco,” Universita di Milano, Milan, Italy; cDivision of Regenerative Medicine, Ospedale San Raffaele Scientific Institute, Milan, Italy Correspondence: Emilio Clementi, M.D., Ph.D., Unit of Clinical Pharmacology, Department of Biomedical and Clinical Sciences, University Hospital “Luigi Sacco,” University of Milano, 20157 Milan, Italy. Telephone: 39-0250319640; Fax: 39-02-50319646; e-mail: emilio.clementi@ unimi.it; or Clara Sciorati, Ph.D, Division of Regenerative Medicine Ospedale San Raffaele Scientific Institute, via Olgettina 58, 20132 Milan, Italy. Telephone: 39-02-26436342; Fax: 39-02-26435283; e-mail: [email protected] Received July 18, 2013; accepted for publication September 5, 2013; first published online in STEM CELLS EXPRESS October 29, 2013. C AlphaMed Press V
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ABSTRACT Duchenne muscular dystrophy (DMD) is an hereditary disease characterized by loss of muscle fibers and their progressive substitution by fat and fibrous tissue. Mesenchymal fibro-adipogenic progenitors (FAPs) expressing the platelet-derived growth factor receptor alpha (PDGFRa) are an important source of fibrosis and adipogenesis in dystrophic skeletal muscle. Among the therapies suggested for dystrophy are those based on nitric oxide (NO) donating drugs, the administration of which slows disease progression. NO has been shown to act by enhancing the regenerative potential of the diseased muscle. Whether it acts also by inhibiting fibrosis and adipogenesis was not known. Here, we show in vitro that NO regulates FAP fate through inhibition of their differentiation into adipocytes. In mdx mice, an animal model of DMD, treatment with the NO donating drug molsidomine reduced the number of PDGFRa1 cells as well as the deposition of both skeletal muscle fat and connective tissues. Inhibition of adipogenesis was due to NO-induced increased expression of miR-27b leading to downregulation of peroxisome proliferator-activated receptors gamma (Pparc1) expression in a pathway independent of cGMP generation. These findings reveal an additional effect of NO in dystrophic muscle that conceivably synergizes with its known effects on regeneration improvement and explain why NO-based therapies appear effective in the treatment of muscular dystrophy. STEM CELLS 2014;32:874–885
INTRODUCTION Duchenne muscular dystrophy (DMD) is a rare disease caused by mutations in the dystrophin encoding-gene, leading to protein loss with an incidence of one out of 3,600–6,000 newborn males [1]. Dystrophin mutations result in skeletal muscle fiber damage and necrosis, leading to progressive substitution of fibers with connective and adipose tissue that leads to death in early adulthood [1]. The pathophysiology of muscular dystrophies also correlates with an altered synthesis of nitric oxide (NO) that is a key signaling molecule controlling adult skeletal muscle structure, bioenergetics, and excitation-contraction coupling [2–5]. In DMD patients, as well as in mouse models of muscular dystrophies such as the mdx mouse, the splice variant of neuronal NO synthase (nNOS) is displaced from the sarcolemma and relocates to the cytosol, an event accompanied by a substantial reduction of enzymatic activity [6–10]. Restoration of NO generation by transgenic expression of nNOS in the mdx mouse model ameliorates its dystrophic phenotype [11, 12] and treatment with
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NO donating drugs reduces muscle necrosis as well as connective tissue deposition [13–16]. We have demonstrated that NO donating drugs administered together with nonsteroidal anti-inflammatory drugs have long-term beneficial effect in mouse models of dystrophy with recovery of skeletal muscle morphology and function [17–19]. The potential of this treatment was explored in a pilot study in dystrophic patients where it showed a high profile of safety and tolerability with promising signs of efficacy [20]. Several groups have explored the mechanisms beyond the therapeutic potential of NO and found that it has multiple actions on survival, activation and differentiation of satellite cells, the mononuclear myogenic precursor cells. These actions enhance the ability of these cells to repair the damaged muscle [21– 26]. Recently, using the NO-donating drug molsidomine, we found that the beneficial effect of NO is explained in part by the ability of the drug to enhance self-renewal of satellite cells, thus delaying the exhaustion of the satellite cells pool and maintaining their regenerative potential [27]. C AlphaMed Press 2013 V
Cordani, Pisa, Pozzi et al. Progressive reduction of muscle regeneration capacity in DMD muscle due to satellite cells exhaustion in DMD correlates with the substitution of degenerated fibers with adipocytes and fibrous tissue. To this substitution contributes significantly a population of mesenchymal fibro-adipogenic progenitors (FAPs) residing in the muscle, specifically located in the interstitial spaces [28–31]. These cells can differentiate efficiently into adipocytes [28, 29], fibroblasts as well as osteoblastic and smooth muscle cells under specific culture conditions [28–30]. While not generating myofibers, they proliferate following muscle damage, fulfilling a trophic support function for myoblasts, to eventually return to a quiescent state in healed muscles [31]. When regeneration fails, however, FAPs generate the components of the fibro-fatty tissue infiltrates that are characteristic of the degenerating muscle [31–33]. Here, we show that long-term treatment of mdx mice in vivo with molsidomine reduces FAP numbers in skeletal muscle tissue and inhibits adipose tissue deposition and indirectly fibrosis. These are direct effects of NO due to a NOdependent regulation of both miR-133a, a known regulator of Collagen 1A1 expression [34] and miR-27b, a key inhibitor of adipocytes differentiation controlling the expression of peroxisome proliferator-activated receptor c (Pparc) [35–37]. In particular, the effect of NO in vitro on adipogenesis was due to cGMP-independent inhibition of Pparc via control of promoter activity and enhancement of expression of miR-27b. These results indicate an action of NO donating drugs additional to the already known effects on muscle myofibers and satellite cells that is also involved in muscle repair. The fact that NO acts by preventing multiple mechanisms of muscle degeneration contributes to explain why NO-donating drugs are effective in dystrophy and suggests that their therapeutic potential should be explored further. In this respect, it is important to note that molsidomine has a good profile of safety and tolerability in humans [38].
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Animals and Treatments C57BL/6J wild-type (WT) mice (Charles River, Wilmington, MA; http://www.criver.com) and mdx24cv mice (B6Ros.CgDmdmdx-4cv/J, C57/BL/6 background) (Jackson Laboratory, Bar Harbor, ME; http://www.jax.org) were treated in accordance with European Community guidelines and with the approval of the Institutional Ethical Committee. Mice were fed either with standard diet or a diet containing 3 mg/kg of molsidomine (1-ethoxy-N-(3-morpholino-5-oxadiazol-3-iumyl) methanimidate, Mucedola S.r.l., Milan, Italy), according to the measured daily food intake. Mdx mice (eight animals per group) were treated starting at 1 month of age and sacrificed 5 months upon treatment for histological and molecular analyses.
Isolation of FAPs For FAP cells isolation, hind limb muscles of 8–10 weeks old mice were minced and digested with 0.2% of Collagenase II (Worthington Biochemical, Lakewood, NJ, http://worthingtonbiochem.com) following published protocols [29]. Cells were stained with the following antibodies: anti-CD31phycoerythrin (PE)/Cy7 (PECy7, clone 390), anti-CD45-PECy7
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(clone 30-F11) (eBioscience, Inc., San Diego, CA, http://www. ebioscience.com), anti-SM/C2.6-Biotin (kindly provided by Dr. Fukada) [39], streptavidin-phycoerythrin (PE, BioLegend, San Diego, CA, http://www.biolegend.com), anti-a7-Integrin-PE (AbLab, UBC Antibody Lab, Vancouver, Canada, http://www. ablab.ca), anti-platelet-derived growth factor receptor alpha (PDGFRa)-allophycocyanin (APC, CD140a clone APA5, BioLegend), anti-LY-6A/E SCA-1-allophycocyanin/Cy7 (APC/Cy7, clone B7, BD Biosciences, Franklin Lakes, NJ, http://www.bdbiosciences.com), and 7-aminoactinomycin D (Life Technologies, Paisley, U.K., http://www.lifetech.com). FAP cells were selected as CD452/ CD312/SM/C2.62 or a7-Integrin2/SCA-11 and PDGFRa1 cells.
Cell Culture Freshly isolated FAP cells were plated at 1 3 104 cells per centimeter square in multiwell plates or in chamber slides and maintained for 6 days in growth medium (GM) consisting of Dulbecco’s modified high glucose Eagle’s medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin plus 5 ng/ml of recombinant human basic fibroblast growth factor (PeproTech, Rocky Hill, NJ; http://www.peprotech.com) in a humidified incubator (37 C and 5% CO2) using plates coated with growth factor reduced BD Matrigel Matrix (BD Biosciences 2350 Qume Drive San Jose, CA http://www.bdbiosciences.com). For differentiation into fibroblasts, FAP cells were cultured for 5 days in DMEM containing 2% FBS and 2 ng/ml of transforming growth factor b 1 (TGFb-1) (Sigma-Aldrich, St. Louis, MO; http://www.sigmaaldrich.com) on eight-well LabTeck Chamber slides (Nunc, Thermo Fisher Scientific Inc Waltham, MA; https://http://www.nuncbrand.com). For adipogenic differentiation, FAP cells were cultured in an adipogenic medium (AM) consisting of DMEM—Ham’s F-12 Nutrient Mixture (DMEM-F-12) with 10% FBS and supplemented with 0.5 mM the phosphodiesterase inhibitor 3isobutyl-1-methylxanthine, 1 mM dexamethasone, and 1 mg/ml insulin (all from Sigma Aldrich). After 3 days in AM, cells were switched to DMEM-F-12 with 10% FBS and 10 mg/ml insulin, which was changed every 2 days. Experiments were carried out using 50 mM of the NO donor 1-[N-(2-aminoethyl)-N-(2ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO, Calbiochem, Merck-Millipore, Frankfurt, Germany; http://www. merckmillipore.com), except for the experiments of Supporting Information Figure S2 where the concentration used were also 10 mM and 20 mM, 10 mM of the guanylate cyclase inhibitor 1H-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, Alexis Biochemicals, Vinci-Biochem S.r.l., FI; http://www.enzolifesciences. com) or vehicle.
Gene and MicroRNA Expression Analysis Total RNA was isolated from cultured cells and homogenized tibialis anterior (TA) muscles with Qiagen RNeasy Micro kit (Qiagen, Hamburg, Germany; http://www.qiagen.com) or the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions, and then reverse-transcribed into cDNA using SuperScript VILO cDNA synthesis kit (Invitrogen- Life Technology, Carlsband, CA http://www.invitrogen.com). Specific sets of primer pairs, as described in Supporting Information Figure 1, were designed to amplify the desired gene sequence. Realtime PCR (RT-PCR) was performed using the SYBR Green Supermix (Biorad, Hercules CA) on a Roche LightCycler 480 C AlphaMed Press 2013 V
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Figure 1. Nitric oxide (NO) downregulates the number of PDGFRa1 cells. (A): Number of PDGFRa1 in tibialis anterior muscle of WT or in mdx mice treated with standard diet (MDX SD) or a diet containing the NO donating drug molsidomine (MDX MOLS): representative images of PDGFRa immuno-staining are also shown (right: 320, scale bars 5 50 mm.). (B): Pdgfra mRNA expression. Data are shown as mean 6 SE; n 4 **, p < .01; ***, p < .001 versus MDX SD. Abbreviations: MOLS, molsidomine or 1-ethoxy-N-(3-morpholino-5-oxadiazol-3-iumyl)methanimidate; PDGFRa, platelet-derived growth factor receptor alpha; WT, wild type.
Instrument (Roche Diagnostics, Rotkreuz, Switzerland; https:// http://www.roche-applied-science.com). All reactions were run in duplicates. Expression levels were analyzed using the 2(2DDCt) method and relative expression levels (arbitrary units, A.U.) using the 2(2DCt) method [40]. Gapdh (for cells) or Rpl32 (for tissues) were used as reference genes to normalize mRNA expression levels. Each plot shown in figures is a mean of at least three independent experiments. RT-PCR-based detection of mature microRNAs (miRNAs) (miR-133a, miR-27b, miR-27a, miR-130a, and miR-181a) and mouse-specific reference snoRNAs (SNO 55 and SNO U6) was carried out from FAPs and tissue samples isolated using the microRNA Reverse Transcription Kit and gene-specific primers and probes (TaqMan MicroRNA Assays), according to the manufacturer’s specifications. All reactions were run in duplicates; snoRNA 55 and snoRNA U6 were used as endogenous genes in tissue and cells analyses, respectively. For these experiments, miRNA expression levels were calculated using the 2(2DCt) formula. All reagents, including primers and kits, were obtained from Applied Biosystems (Life Technologies, Carlsbad, CA). C AlphaMed Press 2013 V
Immunofluorescence Animals were killed by cervical dislocation, TA muscles dissected and immediately frozen in liquid N2-cooled isopentane. Analyses were performed on 10 mm serial sections fixed for 10 minutes in 4% paraformaldehyde. Sections were then blocked 30 minutes with 10% goat serum, 0.1% Triton X-100, incubated overnight with primary antibodies at 4 C followed by appropriate secondary antibodies (Alexa Fluor 488 or 546conjugated; Invitrogen). The following antibodies were used: anti-mouse PDGFRa (R&D System, Minneapolis MN; http:// www.rndsystems.com), anti-mouse PERILIPIN A (Sigma), antimouse LAMININ (Abcam, Cambridge, U.K.; http://www.abcam. com), anti-peroxisome proliferator-activated receptors gamma (PPARc) (E-8, Santa Cruz, CA; http://www.scbt.com), and antimouse COL3A1 (S-17, Santa Cruz). Nuclei were counterstained with Hoechst 33342 (Molecular Probes, Life Technologies) and slides were finally mounted with the ProLong Gold antifade reagent (Invitrogen, Life Technologies). Stained tissues were photographed with a LEICA CTR4000 fluorescence microscope STEM CELLS
Cordani, Pisa, Pozzi et al. LEICA, Allendale NJ, and processed using the ImageJ software (http://rsb.info.nih.gov/ij). FAP cells number was calculated by counting the number of interstitial PDGFRa1 cells per field while fat deposition was measured by calculating PERILIPIN area (mm2) per field in four representative images of four independent animals per group. Immunofluorescence on FAP cells was performed following the protocol described above.
Oil Red O Staining FAP cells were fixed in 4% paraformaldehyde for 10 minutes and then stained for 1 hour by incubating with 0.5% oil red O (Sigma) in a 60:40 isopropanol/water mixture, filtered, and washed twice in distilled water. Nuclei were counterstained with Mayer Hematoxylin and then photographed with a LEICA CTR4000 microscope.
Western Blot Analyses Total lysates were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, and probed using the following antibodies: anti-mouse C-EBPb (C-19, Santa Cruz), anti-mouse Kruppel-like factor 4 (KLF4) (Genespin, Milan, Italy), anti-mouse GADD153/ CHOP (B-3, Santa Cruz), anti-mouse PPARc (H-100x Santa Cruz), anti-mouse C-EBPa (14AA, Santa Cruz), anti-mouse PERILIPIN A (Sigma), and anti-mouse VINCULIN (Sigma). Chemiluminescent HRP-conjugated secondary antibodies were developed using the Super Signal West Femto substrate (Pierce, Thermo Fisher Scientific, Inc., Whaltham, MS) or the Clarity substrate (Biorad) and acquired with a ChemiDoc XRS imager (Bio-Rad). Analyses were performed using the Image J and Image Lab softwares (Biorad). VINCULIN was used to normalize protein expression level.
Luciferase Reporter Assay For reporter analyses, FAP cells were seeded as previously described in 24-well plates. When they reached 80% confluence, cells were transfected with Lipofectamine 2000 (Invitrogen, Life Technologies) following the manufacturer’s instructions. Six hours after transfection, adipogenic differentiation was initiated by AM supplemented with DETA-NO or vehicle. Pparc1 promoter activity was assessed using the pGL3PPARc1 (pGL3-PPARc1 p3000) construct, a gift by Dr. Brune, who kindly provided also and pGL3-PPARc-30 -UTR and pGL3PPARc-30 -UTR-C83A/U84G constructs [35]. The vector pGL3basic was kindly provided by D. Gabellini (Ospedale San Raffele). The latter two constructs were used to test PPARc-30 UTR, where the miR-27 binding site is located. Cells were harvested 3 days after transfection, kept in AM in presence of DETA-NO or vehicle. Firefly luciferase and Renilla activities were measured using the Dual Luciferase Reporter Assay System and the Glo-Max Multi detection System (Promega, Madison, MS; http://www.promega.com), respectively.
Transfections FAP cells were seeded as described above, in six-well plates and transfected when they reached 80% confluence either with 50 nM anti-miR-27b (hsa-miR-27b-3p inhibitor— MIMAT0000126—Ambion, Life Technologies) or 50 nM antimiR inhibitor as a negative control (mirVana miRNA Inhibitor, Negative Control #1, Ambion, Life Technologies), using RNAIMAX (Life Technologies) as per manufacturer’s instructions. Six hours after transfection adipogenic differentiation was started
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by changing medium to AM supplemented either with DETANO or vehicle as a control. Transfected cells were harvested and analyzed 6 days later.
Statistical Analyses All values are expressed as mean 6 SEM of at least three independent experiments. All data were analyzed using either an unpaired two-tailed Student’s t test for comparisons between two groups, and one-way ANOVA with appropriate post-test adjustment for multiple group comparisons. p-Values lower than 5% (p < .05) were considered statistically significant, single, double, triple, and quadruple asterisks in the figures indicate p < .05, p < .01, p < .001, and p < .0001, respectively.
RESULTS NO Decreases the Number of PDGFRa1 Cells in Dystrophic Muscle We evaluated the effect on FAP number in dystrophic muscle of oral administration of molsidomine for 5 months to mdx mice. PDGFRa1 cells were barely detectable in TA of WT animals and abundant in mdx mice treated with standard diet. Molsidomine treatment significantly decreased the number of PDGFRa1 cells (Fig. 1A). RT-PCR analyses confirmed these data by showing a 3.5-fold decrease of Pdgfra expression in mdx mice treated with molsidomine compared to those treated with the standard diet (Fig. 1B).
Collagen 1A1 and 3A1 Are Reduced in NO-Treated Mdx Muscle PDGFRa1 cells are abundant in fibrotic muscle and express fibrosis-related markers [30]. We investigated the expression of Collagen 1A1 (Col1A1) and Collagen 3A1 (Col3A1), two markers of fibrosis, in TA muscles. Mdx mice fed with standard diet expressed high levels of both collagen isoforms in comparison to WT mice, while the 5 months treatments with molsidomine treatment reduced significantly the levels of both mRNAs (Fig. 2A). Consistently with these data, mdx mice showed reduced levels of miR-133a, a known negative regulator of Col1A1 [34], which was increased by molsidomine treatment (Fig. 2B).
NO Reduces Fat Deposition in Dystrophic Mice We evaluated fat deposition in mdx mice by assessing the levels of PERILIPIN, a protein coating lipid droplets of adipocytes [41]. As reported in Figure 3A, PERILIPIN positivity was significantly increased in mdx mice with respect to WT mice and normalized after treatment with molsidomine. To evaluate better the inhibitory effect of NO on fat deposition, we measured the expression of two biomarkers of mature adipocytes, the fatty acid binding protein 4 (Fabp4) and Adiponectin, and of two adipose-regulating transcription factors, Pparc1 and the CCAAT/enhancer-binding protein a (C-Ebpa). Pparc1, C-Ebpa, Fabp4, and Adiponectin mRNA expression levels in TA of mice treated for 5 months with molsidomine were almost halved when compared with mice that received standard diet, and comparable to those of WT mice (Fig. 3B). Consistently with these data, the expression of miR-27b a negative regulator of Pparc that is abundantly expressed in WT mice was significantly reduced in standard diet-receiving mice and reinduced in molsidomine-treated animals (Fig. 3C). C AlphaMed Press 2013 V
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Figure 2. Nitric oxide (NO) inhibits collagen deposition. (A): Collagen 1A1/3A1 mRNA expression and (B) miR-133a expression in muscle of WT or mdx mice treated with standard diet (MDX SD) or a diet containing the NO donating drug molsidomine (MDX MOLS). Data are shown as mean 6 SE; n 5. *, p < .05; ***, p < .001 versus MDX SD. Abbreviations: MOLS, molsidomine or 1-ethoxy-N-(3-morpholino-5oxadiazol-3-iumyl)methanimidate; WT, wild type.
NO Inhibits Differentiation of FAPs into the Adipogenic Lineage While Not Affecting Their Differentiation into Collagen-Producing Cells We investigated whether inhibition of collagen and fat deposition induced by NO donation in vivo can be ascribed to regulation of FAP fate. We purified from the hind limb muscles of WT and mdx mice FAPs, that were defined as mesenchymal cells that are CD452 (hematopoietic lineage negative), CD312 (endothelial negative), SMC2.62 or a7 integrin2 (satellite cells negative), and SCA-11/PDGFRa1 cells [28, 29]. FAPs were subsequently cultured in a profibrotic medium containing TGFb1 [30], either in the presence of the NO donor DETA-NO or vehicle. Immunofluorescence with anti-COL3A1 antibody was carried out to evaluate the effects of NO on differentiation of matrix-producing cells. The percentage of collagen1 cells remained unchanged after DETA-NO treatment when compared with vehicle both in FAPs isolated from WT and mdx mice (Fig. 4A). To investigate the effect of NO donation on FAPs adipogenic potential, we performed oil red O dye staining on WTand mdx-derived FAPs cultured under adipogenic conditions using a specific induction medium (AM). DETA-NO strongly decreased the number of Red Oil1 cells both in WT and mdxderived FAPs cells with respect to vehicle suggesting that DETA-NO drastically affects FAPs adipogenic potential (Fig. 4B). The effect of DETA-NO was concentration dependent (Supporting Information Fig. S2).
NO Inhibits FAPs Adipogenic Potential in a cGMP-Independent Way To investigate the mechanism of NO-induced inhibition of FAPs differentiation into adipocytes, we first focused our attention on the expression of the transcription factors directC AlphaMed Press 2013 V
ing the adipogenic lineage. DETA-NO did not modify KLF4, CEBPb, and CHOP/GADD153 protein levels early after adipogenesis induction (Supporting Information Fig. S3), while it significantly inhibited PPARc, a transcription factor present in committed preadipocytes and mature cells [42], and reduced the expression of PERILIPIN (Fig. 5A). NO acts via generation of cyclic GMP (cGMP) and/or via cGMP-independent mechanisms. To assess the cGMP dependency of the NO action, we induced FAPs differentiation in AM in presence of DETA-NO, either alone or in combination with the guanylate-cyclase inhibitor ODQ. The inhibitory effect of DETA-NO on the mRNA expression levels of Pparc1, C-Ebpa, Fabp4, and Adiponectin was not reversed by ODQ, which per se did not have any effect (Fig. 5B). Likewise, ODQ did not modify the effect of DETA-NO on PPARc1, C-EBPa, fatty acid binding protein 4, and PERILIPIN protein levels (Fig. 5C).
NO Regulates PPARc Via MiR-27b We investigated how NO inhibits Pparc expression. To this end, we analyzed the TF promoter and its transcript regulation. We performed luciferase reporter assays in FAPs transiently transfected with a construct containing the luciferase encoding region under the control of the Pparc promoter 1(PGL3-c1) [35]. After transfection, FAPs cells were cultured in AM containing DETA-NO or vehicle. AM induced a significant increase in Pparc promoter 1 versus GM (Supporting Information Fig. S4). A 40% reduction of activity was detected in the presence of DETA-NO (Fig. 6A). Given the greater reduction of Pparc1 mRNA levels induced by DETA-NO in comparison with Pparc promoter activity downregulation (Fig. 5B vs. Fig. 6A), we tested the hypothesis that an additional posttranscriptional regulation of Pparc1 by NO was present. We measured the activity of both WT PPARc-30 -UTR and mutated STEM CELLS
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Figure 3. Nitric oxide (NO) inhibits fat deposition. (A): PERILIPIN staining in WT or mdx mice treated with standard diet (MDX SD) or a diet containing the NO donating drug molsidomine (MDX MOLS). Upper panel shows data quantification as PERILIPIN1 area (mean6 SE; n 5 7); lower panels show representative images of PERILIPIN immuno-staining (320, scale bars 5 50 mm). (B): Pparc1, C-ebpa, Fabp4, and Adiponectin mRNA and (C) miR-27b expression (mean 6 SE, n 3, *, p < .05; **, p < .01; ***, p < .001 vs. MDX SD). Abbreviations: MOLS, molsidomine or 1-ethoxy-N-(3-morpholino-5-oxadiazol-3-iumyl)methanimidate; WT, wild type.
30 -UTR containing vectors normalized versus pGL3-basic in transiently transfected FAPs cultured in AM. DETA-NO reduced significantly the luciferase activity of the PPARc-30 -UTR but was ineffective when miR-27 binding site was mutated (Fig.
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6B). When we examined the expression of miRNAs possibly implicated in adipogenic differentiation, namely miR-27a/ 130a/181a and miR-27b, we found that only miR-27b was affected by DETA-NO treatment (Fig. 6C and Supporting C AlphaMed Press 2013 V
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Figure 4. NO modulates in vitro fibro-adipogenic progenitors (FAPs) adipogenic potential but not FAP differentiation into collagenexpressing cells. (A): Data quantification (left) and representative images (320, scale bars 5 50 mm; right) of COLLAGEN 3A1 staining in TGFb-treated FAPs isolated from WT or mdx mice in the presence of the NO donor DETA-NO or vehicle. (B): Oil red O-staining in FAPs isolated and treated as described above and cultured in adipogenic conditions. Data quantification is in the upper panel while representative images of the staining (310, scale bars 5 100 mm or 340 magnification of the boxes, scale bars 5 25 mm) in the lower panels. Data are expressed as mean 6 SE; n 5, ***, p < .001; ****, p < .0001 versus vehicle. Abbreviations: DETA-NO, DETA-NONOate (1-[N-(2aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate); WT, wild type.
Information Fig. S5). In particular, miR-27b expression levels were high in proliferating FAPs (GM) and reduced by almost threefold when adipogenesis was induced by switch to AM. DETA-NO increased twofold the expression of miR-27b when compared with vehicle. To confirm these data further, we evaluated the effect of DETA-NO treatment on Pparc1 mRNA expression level after transient transfection with antagomiR27b. As shown in Figure 6D, miR-27b was silenced by its own C AlphaMed Press 2013 V
antagomiR with respect to the negative control (a-miR-27b vs. a-miR neg, upper panel). NO-induced Pparc1 inhibition completely disappeared when antagomiR-27b was present.
DISCUSSION Adult skeletal muscle regenerates upon damage thanks to the ability of resident proliferating satellite cells capable of forming STEM CELLS
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Figure 5. NO inhibits fibro-adipogenic progenitors (FAPs) adipogenic differentiation by a cGMP-independent mechanism. (A): Representative images of PPARc and PERILIPIN immuno-staining in FAPs treated with DETA-NO or vehicle (320, scale bars 5 50 mm). (B): Expression of the mRNA of adipogenesis-markers (Pparc1, C-Ebpa, Fabp4, and Adiponectin) in FAPs treated with DETA-NO, the guanylatecyclase inhibitor ODQ, or vehicle. (C): Representative images (upper panels) and quantification (lower panels) of adipogenesis-markers (PPARc1, C-EBPa, and PERILIPIN) protein expression in FAPs treated as above. VINCULIN was used as an internal control. Data are presented as fold change to vehicle-treated cells (fixed at 1) and reported as mean 6 SE, n 4. *, p < .05; **, p < .01; ***, p < .001 versus vehicle. Abbreviations: DETA-NO, DETA-NONOate (1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate); PPARc, peroxisome proliferator-activated receptors gamma.
new fibers. When chronic damage occurs, such as in dystrophy, and compensative regeneration fails because of progressive exhaustion of the satellite cells pool [43–45], fibro-adipogenic degeneration becomes predominant, in a process termed “reparative disorder” [33]. An important role in this process has been recently demonstrated for a group of mesenchymal stem cells, FAPs, capable of differentiating in vitro toward both fibrogenic and adipogenic lineages [31]. FAPs are located into the interstitial space between myofibers close to vessels and can be purified as CD452, CD312, a7-Integrin2, CD341, Sca11, and PDGFRa1 cells [28, 29]. Upon damage FAPs expand,
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prior to satellite cells proliferation, and invade the surrounding tissue. Subsequently, FAPs are either cleansed from the invaded tissue (in case of a successful repair process) or, if the regeneration process is defective or continuous as in degenerative muscle disorders, they differentiate into adipocytes [46]. Investigation of the cellular and molecular effectors of the regeneration/degeneration switch is relatively recent and pathways and regulatory mechanisms still need to be identified in full [47, 48]. The role of these cells and their regulation appears to be important in the overall process of muscle healing alongside the much better characterized pathogenesis of skeletal muscle C AlphaMed Press 2013 V
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Figure 6. NO regulates Pparc promoter and affects its mRNA stability controlling miR-27b. (A): Luciferase reporter assay in fibroadipogenic progenitors (FAPs) transfected with the Pparc1 promoter construct or (B) with constructs of normal (pGL3-PPARc-30 -UTR) or mutated (pGL3-PPARc-30 -UTR-C83A/U84) 30 -UTR of Pparc promoter region and treated with DETA-NO or vehicle. Data are expressed as mean 6 SE after normalization to pGL3 basal activity. (C): Mir-27b expression in FAPs cultured in proliferating (GM) or in adipogenic conditions (AM) and treated with DETA-NO or vehicle. (D): MiR-27b (upper) or Pparc1 (lower) expression in FAPs cultured in adipogenic conditions in the presence or absence of DETA-NO and transfected with anti-miR-27b (a-miR-27b) or negative control (a-miR neg). Data are presented as mean of relative expression 6 SE, n 3. *, p < .05; **, p < .01 versus vehicle or versus DETA-NO 1 a-miR neg. for panel (D). Abbreviations: DETA-NO, DETA-NONOate (1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate); PPARc, peroxisome proliferator-activated receptors gamma.
disease and interesting novel therapeutic targets. The shedding of light on FAPs, especially considering the complexity of skeletal muscle cells network interactions, is thus crucial as it would add novel therapeutic options to muscular dystrophy. Here, we show that oral administration of the NO donating drug molsidomine in mdx mice decreases the number of FAPs in muscle while reducing collagen expression as well as adipose tissue deposition. We analyzed the mechanism of NO action. NO delivered with the NO donor DETA-NO inhibited the expression of the C AlphaMed Press 2013 V
adipocyte markers PERILIPIN, Adiponectin, and Fabp4 as well as adipogenic differentiation by both mdx and WT-derived FAPs. Interestingly, the action of NO was not exerted at the initial stages of adipogenic differentiation, as it affected expression only of the middle adipogenic transcription factors Pparc and C-ebpa that coordinate and direct the development of adipogenic lineage [42]. NO did not affect, in the experimental condition used, expression of the early adipogenesis transcription factors KLF4, c-EBPb, and CHOP10 while regulating PPARc1, a key transcription factor in adipogenesis [42, 49, 50]. PPARc is a STEM CELLS
Cordani, Pisa, Pozzi et al. member of the nuclear hormone receptors subfamily, highly enriched in adipose tissue. PPARc ectopic expression and activation in fibroblasts promote their conversion into lipid-filled adipocytes inducing a complete adipogenic gene expression program [51]. In particular, we focused on Pparc1 since it is able to drive in itself development of adipose tissue in mouse independently of Pparc2 [52]. We unequivocally demonstrate that NO regulates Pparc1 expression by regulating the expression of miR-27b, a specific adipogenesis-regulator that in turn controls Pparc mRNA stability [36, 37]. MicroRNAs (miRNAs) are small non-protein coding RNAs, some of which acting as post-transcriptional gene regulators in muscle development and function [53]; their expression is altered in several muscle disorders including DMD [54, 55]. No specific information about the function and regulation of miR-27b in DMD and in adipogenesis regulation in muscle however was until now available. Our results and in particular the observation that induction of miR-27b by NO occurred both in vitro (isolated FAPs) and in vivo (mdx mice) suggest that miR-27b may be the mechanism by which NO endogenously generated by myofibers reduces adipogenesis and fat tissue deposition in muscular dystrophy. We also found that NO enhances the expression of miR133a, the expression of which we found to be reduced in mdx muscles as already demonstrated [56]. We cannot at present identify a specific functional role of this inhibitory action by NO. The role of miR-133a, whose levels are lower in sera from DMD patients when compared with healthy individuals [56], is not yet clear as it seems to be dispensable for normal muscle development [57] although regulating Col1A1 synthesis in several tissues. We have not analyzed in detail how NO regulates miR-27b expression except for the cGMP-independency of its effect. Interestingly, alongside miR-133a, NO regulates also other miRNAs in skeletal muscle [56, 58, 59]. In particular, a single local injection in mdx muscle of the NO donating drug nitroglycerin increased the expression of miR-1 and miR-29. This was due to S-nitrosylation [60] and then removal of the inhibitory histone deacetylase 2 from the promoters of these miRNA [56]. Whether regulation of miRNAs 133a and 27b is mediated by the same mechanism remains to be investigated further. Interestingly, miR-1 is linked to the redox state of cell, and miR-29 is involved into the fibrotic process [56]. Our observation that NO also controls adipogenic differentiation via miR-27b indicates that this is a relevant mechanism by which NO controls muscle homeostasis. How this integrates with the other wellknown homeostatic actions of NO, for example, on muscle bioenergetics, glucose uptake, and excitation contraction coupling remains also to be investigated [5, 26, 61–63]. We found that molsidomine in vivo reduces collagen expression in mdx mice, while DETA-NO in vitro did not influence TGFb-induced FAPs differentiation into fibroblasts. This suggests the possibility that extracellular matrix producing cells other than FAPs are regulated by NO. Fibrocytes, and in particular myofibroblasts, are an important source of extracellular matrix deposition in dystrophic muscle. These cells are present during tissue repair and their activation persists because of constant myofibers breakdown and chronic inflammation [31, 64, 65].
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We have recently demonstrated using a-sarcoglycan null mice, another animal model of muscular dystrophy, that molsidomine is endowed with therapeutic effects, maintaining the functional pool of satellite cells and stimulating the regeneration capacity of skeletal muscle [27]. Moreover, molsidomine modulates the characteristics of the inflammatory cell infiltrate within a-sarcoglycan null dystrophic muscles, and this contributes to its healing function. In particular, molsidomine boosts the subset of macrophages associated to reduced fibrosis and increased preservation of the muscle tissue [66]. The combination of these different but synergic actions of molsidomine on muscle repair together with the good profile of safety and tolerability [20] are in keeping with the idea that molsidomine and other NO donating drugs are precious tools for DMD patients therapy that need to be investigated and refined further.
CONCLUSIONS This work demonstrates for the first time both in vivo and in vitro that NO regulates FAPs number and fate controlling fat tissue deposition in dystrophic skeletal muscle. The underlying mechanism of these actions is regulation of Pparc1 expression through direct modulation of the promoter activity and posttranscriptional control of miR-27b levels in a cGMPindependent way. Moreover, NO inhibits collagen deposition affecting FAPs in vivo. Altogether, these data point out the therapeutic role of NO donating drugs in the treatment of skeletal muscle dystrophy in virtue of their ability to affect multiple mediators playing a role in the complex pathogenic process of the disease.
ACKNOWLEDGMENTS We thank Prof. Bernard Br€ une (Goethe-University Frankfurt) for providing to us the pGL3-PPARc and related vectors. We are grateful to Prof. So-ichiro Fukada (Osaka University, Osaka, Japan) for the SM/C2.6 antibody. We thank Dr. Silvia Brunelli (University of Milano-Bicocca, Milan, Italy) for helpful discussion. This work was supported by the European Community’s framework programme FP7/2007–2013 under grant agreement n 241440 (ENDOSTEM) (to E.C.), the Italian Ministry of Health RC 2013 (to E.C.), Associazione Italiana Ricerca sul Cancro (AIRC IG11362) (to E.C.), and from the Ministero della Universita e Ricerca PRIN 2010–2011 (to E.C.).
AUTHORS CONTRIBUTIONS N.C.: conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing; V.P.: collection and assembly of data and data analysis and interpretation; L.P.: collection and assembly of data; C.S.: conception and design and manuscript writing; E.C.: conception and design, manuscript writing, financial support, and final approval of the manuscript. C.S. and E.C. are co-corresponding authors.
DISCLOSURE
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POTENTIAL CONFLICTS
OF INTEREST
The authors indicate no potential conflicts of interest.
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