A nitric oxide synthase transgene ameliorates muscular dystrophy in mdx mice

Dystrophin-deficient muscles experience large reductions in expression of nitric oxide synthase (NOS), which suggests that NO deficiency may influence the dystrophic pathology. Because NO can function as an antiinflammatory and cytoprotective molecule, we propose that the loss of NOS from dystrophic muscle exacerbates muscle inflammation and fiber damage by inflammatory cells. Analysis of transgenic mdx mice that were null mutants for dystrophin, but expressed normal levels of NO in muscle, showed that the normalization of NO production caused large reductions in macrophage concentrations in the mdx muscle. Expression of the NOS transgene in mdx muscle also prevented the majority of muscle membrane injury that is detectable in vivo, and resulted in large decreases in serum creatine kinase concentrations. Furthermore, our data show that mdx muscle macrophages are cytolytic at concentrations that occur in dystrophic, NOS-deficient muscle, but are not cytolytic at concentrations that occur in dystrophic mice that express the NOS transgene in muscle. Finally, our data show that antibody depletions of macrophages from mdx mice cause significant reductions in muscle membrane injury. Together, these findings indicate that macrophages promote injury of dystrophin-deficient muscle, and the loss of normal levels of NO production by dystrophic muscle exacerbates inflammation and membrane injury in muscular dystrophy

Targeting a therapeutic LIF transgene to muscle via the immune system ameliorates muscular dystrophy.

Many potentially therapeutic molecules have been identified for treating Duchenne muscular dystrophy. However, targeting those molecules only to sites of active pathology is an obstacle to their clinical use. Because dystrophic muscles become extensively inflamed, we tested whether expressing a therapeutic transgene in leukocyte progenitors that invade muscle would provide selective, timely delivery to diseased muscle. We designed a transgene in which leukemia inhibitory factor (LIF) is under control of a leukocyte-specific promoter and transplanted transgenic cells into dystrophic mice. Transplantation diminishes pathology, reduces Th2 cytokines in muscle and biases macrophages away from a CD163+/CD206+ phenotype that promotes fibrosis. Transgenic cells also abrogate TGFβ signaling, reduce fibro/adipogenic progenitor cells and reduce fibrogenesis of muscle cells. These findings indicate that leukocytes expressing a LIF transgene reduce fibrosis by suppressing type 2 immunity and highlight a novel application by which immune cells can be genetically modified as potential therapeutics to treat muscle disease

Ultrastructural modifications of myotendinous junction with disuse

The skeletal muscle contraction is transmitted to the tendon through the myotendinous junction (MTJ), where the proximal extremity of tendon forms characteristic finger-like processes, penetrating into the muscle mass (Ciena et al., 2010). MTJ represents an intriguing and complex structure from both biological and functional point of view. We recently demonstrated that changes at MTJ level occur as an adaptation to exercise-induced tension increase. In particular, branching of finger-like processes increases, so enlarging the whole tendon-muscle surface area and, consequently, allowing a better tension resistance (Curzi et al., 2012). The aim of this study is to analyse MTJ behavior in disuse condition, furtherly investigating the strict morphofunctional correlation. Therefore, 4 hind-limb suspended and 4 control rats were studied. After sacrifice, MTJs of plantaris muscle were withdrawn, fixed in 1.4% glutaraldehyde in 0.2M sodium cacodylate and processed as previously described (Tidball et al., 1992; D’Emilio et al., 2010). After 5 days of suspension, skeletal muscle highlights signs of atrophy in response to decreased mechanical loading (Linderman et al., 1994). In fact, close to muscle-tendon interface, irregular misaligned sarcomeres, with absent Z-lines, were observed. Muscle intermyofibrillar component spreads, especially between terminal filaments and tendon finger-like processes. A lower number of muscle-tendon interdigitations, characteristically not bifurcated, also appears. In conclusion, along with muscle atrophy features, ultrastructural changes occur at the MTJ organization level, as an adaptation to muscle unloading

Differential Effects of Myeloid Cell PPARδ and IL-10 in Regulating Macrophage Recruitment, Phenotype, and Regeneration following Acute Muscle Injury

Changes in macrophage phenotype in injured muscle profoundly influence regeneration. In particular, the shift of macrophages from a pro-inflammatory (M1-biased) phenotype to a pro-regenerative (M2-biased) phenotype characterized by expression of CD206 and CD163 is essential for normal repair. According to the current canonical mechanism regulating for M1/M2 phenotype transition, signaling through PPARδ is necessary for obtaining the M2-biased phenotype. Our findings confirm that the murine myeloid cell targeted deletion of Ppard reduces expression in vitro of genes that are activated in M2-biased macrophages; however, the mutation in mice in vivo increased numbers of CD206+ M2-biased macrophages and did not reduce the expression of phenotypic markers of M2-biased macrophages in regenerating muscle. Nevertheless, the mutation impaired CCL2-mediated chemotaxis of macrophages and slowed revascularization of injured muscle. In contrast, null mutation of IL10 diminished M2-biased macrophages but produced no defects in muscle revascularization. Our results provide two significant findings. First, they illustrate that mechanisms that regulate macrophage phenotype transitions in vitro are not always predictive of mechanisms that are most important in vivo. Second, they show that mechanisms that regulate macrophage phenotype transitions differ in different in vivo environments

Modulation of Klotho expression in injured muscle perturbs Wnt signalling and influences the rate of muscle growth

Skeletal muscle injuries activate a complex programme of myogenesis that can restore normal muscle structure. We tested whether modulating the expression of klotho influenced the response of mouse muscles to acute injury. Our findings show that klotho expression in muscle declines at 3 days post‐injury. That reduction in klotho expression coincided with elevated expression of targets of Wnt signalling (Ccnd1; Myc) and increased MyoD+ muscle cell numbers, reflecting the onset of myogenic cell differentiation. klotho expression subsequently increased at 7 days post‐injury with elevated expression occurring primarily in inflammatory lesions, which was accompanied by reduced expression of Wnt target genes (Ccnd1: 91%; Myc: 96%). Introduction of a klotho transgene maintained high levels of klotho expression over the course of muscle repair and attenuated the increases in Ccnd1 and Myc expression that occurred at 3 days post‐injury. Correspondingly, transgene expression reduced Wnt signalling in Pax7+ cells, reflected by reductions in Pax7+ cells expressing active β‐catenin, and reduced the numbers of MyoD+ cells at 3 days post‐injury. At 21 days post‐injury, muscles in klotho transgenic mice showed increased Pax7+ and decreased myogenin+ cell densities and large increases in myofibre size. Likewise, treating myogenic cells in vitro with Klotho reduced Myod expression but did not affect Pax7 expression. Muscle inflammation was only slightly modulated by increased klotho expression, initially reducing the expression of M2‐biased macrophage markers Cd163 and Cd206 at 3 days post‐injury and later increasing the expression of pan‐macrophage marker F480 and Cd68 at 21 days post‐injury. Collectively, our study shows that Klotho modulates myogenesis and that increased expression accelerates muscle growth after injury

Growth hormone and resistance exercise effects on myotendinous junction in hind-limb unloaded rats: an ultrastructural study

Myotendinous junction (MTJ), is the site at which the contractile forces are transmitted from myofibrils to extracellular matrix, and, when observed at ultrastructural level, it displays an interdigitated profile (Kojima et al., 2008). The aim of this study is to investigate the junctional behaviour in the atrophic condition and during particular prevention protocols. The MTJs of plantaris muscles from twenty hypophysectomized rats were processed for electron microscopy (Curzi et al., 2012). The animals were assigned to one of five groups: control (CTRL), hind-limb suspended (HS), hind-limb suspended and exercised (EX), hind-limb suspended and growth hormone injected (GH) and hind-limb suspended, GH injected and exercised (GH+EX). After unloading, the tendon finger-like processes appeared small and irregular and the contact between tissues is reduced to 61.3%. The prevention treatments increased the interface area up to 6.2% in GH, 25.3% in EX and 46.4 % in GH+EX respectively. The resistance exercise protocol, as well as GH treatment, was not capable of maintaining the contact surface between tissues, but in both exercised groups the number of bifurcated interdigitations was higher than in the CTRL. In conclusion, ultrastructural changes occur at MTJ in HS, as an adaptation to muscle unloading. Differently, MTJ structure is partially maintained by resistance training or GH treatment, while the exercise with simultaneous somatropin administration showed a greater effect

Arginine Metabolism by Macrophages Promotes Cardiac and Muscle Fibrosis in mdx Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is the most common, lethal disease of childhood. One of 3500 new-born males suffers from this universally-lethal disease. Other than the use of corticosteroids, little is available to affect the relentless progress of the disease, leading many families to use dietary supplements in hopes of reducing the progression or severity of muscle wasting. Arginine is commonly used as a dietary supplement and its use has been reported to have beneficial effects following short-term administration to mdx mice, a genetic model of DMD. However, the long-term effects of arginine supplementation are unknown. This lack of knowledge about the long-term effects of increased arginine metabolism is important because elevated arginine metabolism can increase tissue fibrosis, and increased fibrosis of skeletal muscles and the heart is an important and potentially life-threatening feature of DMD.We use both genetic and nutritional manipulations to test whether changes in arginase metabolism promote fibrosis and increase pathology in mdx mice. Our findings show that fibrotic lesions in mdx muscle are enriched with arginase-2-expressing macrophages and that muscle macrophages stimulated with cytokines that activate the M2 phenotype show elevated arginase activity and expression. We generated a line of arginase-2-null mutant mdx mice and found that the mutation reduced fibrosis in muscles of 18-month-old mdx mice, and reduced kyphosis that is attributable to muscle fibrosis. We also observed that dietary supplementation with arginine for 17-months increased mdx muscle fibrosis. In contrast, arginine-2 mutation did not reduce cardiac fibrosis or affect cardiac function assessed by echocardiography, although 17-months of dietary supplementation with arginine increased cardiac fibrosis. Long-term arginine treatments did not decrease matrix metalloproteinase-2 or -9 or increase the expression of utrophin, which have been reported as beneficial effects of short-term treatments.Our findings demonstrate that arginine metabolism by arginase promotes fibrosis of muscle in muscular dystrophy and contributes to kyphosis. Our findings also show that long-term, dietary supplementation with arginine exacerbates fibrosis of dystrophic heart and muscles. Thus, commonly-practiced dietary supplementation with arginine by DMD patients has potential risk for increasing pathology when performed for long periods, despite reports of benefits acquired with short-term supplementation

Neuronal Nitric Oxide Synthase-Rescue of Dystrophin/Utrophin Double Knockout Mice does not Require nNOS Localization to the Cell Membrane

Survival of dystrophin/utrophin double-knockout (dko) mice was increased by muscle-specific expression of a neuronal nitric oxide synthase (nNOS) transgene. Dko mice expressing the transgene (nNOS TG+/dko) experienced delayed onset of mortality and increased life-span. The nNOS TG+/dko mice demonstrated a significant decrease in the concentration of CD163+, M2c macrophages that can express arginase and promote fibrosis. The decrease in M2c macrophages was associated with a significant reduction in fibrosis of heart, diaphragm and hindlimb muscles of nNOS TG+/dko mice. The nNOS transgene had no effect on the concentration of cytolytic, CD68+, M1 macrophages. Accordingly, we did not observe any change in the extent of muscle fiber lysis in the nNOS TG+/dko mice. These findings show that nNOS/NO (nitric oxide)-mediated decreases in M2c macrophages lead to a reduction in the muscle fibrosis that is associated with increased mortality in mice lacking dystrophin and utrophin. Interestingly, the dramatic and beneficial effects of the nNOS transgene were not attributable to localization of nNOS protein at the cell membrane. We did not detect any nNOS protein at the sarcolemma in nNOS TG+/dko muscles. This important observation shows that sarcolemmal localization is not necessary for nNOS to have beneficial effects in dystrophic tissue and the presence of nNOS in the cytosol of dystrophic muscle fibers can ameliorate the pathology and most importantly, significantly increase life-span

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Ten-year mortality, disease progression, and treatment-related side effects in men with localised prostate cancer from the ProtecT randomised controlled trial according to treatment received

Background The ProtecT trial reported intention-to-treat analysis of men with localised prostate cancer randomly allocated to active monitoring (AM), radical prostatectomy, and external beam radiotherapy. Objective To report outcomes according to treatment received in men in randomised and treatment choice cohorts. Design, setting, and participants This study focuses on secondary care. Men with clinically localised prostate cancer at one of nine UK centres were invited to participate in the treatment trial comparing AM, radical prostatectomy, and radiotherapy. Intervention Two cohorts included 1643 men who agreed to be randomised and 997 who declined randomisation and chose treatment. Outcome measurements and statistical analysis Analysis was carried out to assess mortality, metastasis and progression and health-related quality of life impacts on urinary, bowel, and sexual function using patient-reported outcome measures. Analysis was based on comparisons between groups defined by treatment received for both randomised and treatment choice cohorts in turn, with pooled estimates of intervention effect obtained using meta-analysis. Differences were estimated with adjustment for known prognostic factors using propensity scores. Results and limitations According to treatment received, more men receiving AM died of PCa (AM 1.85%, surgery 0.67%, radiotherapy 0.73%), whilst this difference remained consistent with chance in the randomised cohort (p = 0.08); stronger evidence was found in the exploratory analyses (randomised plus choice cohort) when AM was compared with the combined radical treatment group (p = 0.003). There was also strong evidence that metastasis (AM 5.6%, surgery 2.4%, radiotherapy 2.7%) and disease progression (AM 20.35%, surgery 5.87%, radiotherapy 6.62%) were more common in the AM group. Compared with AM, there were higher risks of sexual dysfunction (95% at 6 mo) and urinary incontinence (55% at 6 mo) after surgery, and of sexual dysfunction (88% at 6 mo) and bowel dysfunction (5% at 6 mo) after radiotherapy. The key limitations are the potential for bias when comparing groups defined by treatment received and changes in the protocol for AM during the lengthy follow-up required in trials of screen-detected PCa. Conclusions Analyses according to treatment received showed increased rates of disease-related events and lower rates of patient-reported harms in men managed by AM compared with men managed by radical treatment, and stronger evidence of greater PCa mortality in the AM group. Patient summary More than 95 out of every 100 men with low or intermediate risk localised prostate cancer do not die of prostate cancer within 10 yr, irrespective of whether treatment is by means of monitoring, surgery, or radiotherapy. Side effects on sexual and bladder function are better after active monitoring, but the risks of spreading of prostate cancer are more common