Controlled Delivery of Sdf-1 Alpha and Igf-1: Cxcr4(+) Cell Recruitment and Functional Skeletal Muscle Recovery

Therapeutic delivery of regeneration-promoting biological factors directly to the site of injury has demonstrated its efficacy in various injury models. Several reports describe improved tissue regeneration following local injection of tissue specific growth factors, cytokines and chemokines. Evidence exists that combined cytokine/growth factor treatment is superior for optimizing tissue repair by targeting different aspects of the regeneration response. The purpose of this study was to evaluate the therapeutic potential of the controlled delivery of stromal cell-derived factor-1alpha (SDF-1 alpha) alone or in combination with insulin-like growth factor-I (SDF-1 alpha/IGF-I) for the treatment of tourniquet-induced ischemia/reperfusion injury (TK-I/R) of skeletal muscle. We hypothesized that SDF-1 alpha will promote sustained stem cell recruitment to the site of muscle injury, while IGF-I will induce progenitor cell differentiation to effectively restore muscle contractile function after TK-I/R injury while concurrently reducing apoptosis. Utilizing a novel poly-ethylene glycol PEGylated fibrin gel matrix (PEG-Fib), we incorporated SDF-1 alpha alone (PEG-Fib/SDF-1 alpha) or in combination with IGF-I (PEG-Fib/SDF-1 alpha/IGF-I) for controlled release at the site of acute muscle injury. Despite enhanced cell recruitment and revascularization of the regenerating muscle after SDF-1 alpha treatment, functional analysis showed no benefit from PEG-Fib/SDF-1 alpha therapy, while dual delivery of PEG-Fib/SDF-1 alpha/IGF-I resulted in IGF-I-mediated improvement of maximal force recovery and SDF-1 alpha-driven in vivo neovasculogenesis. Histological data supported functional data, as well as highlighted the important differences in the regeneration process among treatment groups. This study provides evidence that while revascularization may be necessary for maximizing muscle force recovery, without modulation of other effects of inflammation it is insufficient.Kinesiology and Health Educatio

Functional muscle hypertrophy by increased insulin-like growth factor 1 does not require dysferlin.

IntroductionDysferlin loss-of-function mutations cause muscular dystrophy, accompanied by impaired membrane repair and muscle weakness. Growth promoting strategies including insulin-like growth factor 1 (IGF-1) could provide benefit but may cause strength loss or be ineffective. The objective of this study was to determine whether locally increased IGF-1 promotes functional muscle hypertrophy in dysferlin-null (Dysf-/- ) mice.MethodsMuscle-specific transgenic expression and postnatal viral delivery of Igf1 were used in Dysf-/- and control mice. Increased IGF-1 levels were confirmed by enzyme-linked immunosorbent assay. Testing for skeletal muscle mass and function was performed in male and female mice.ResultsMuscle hypertrophy occurred in response to increased IGF-1 in mice with and without dysferlin. Male mice showed a more robust response compared with females. Increased IGF-1 did not cause loss of force per cross-sectional area in Dysf-/- muscles.DiscussionWe conclude that increased local IGF-1 promotes functional hypertrophy when dysferlin is absent and reestablishes IGF-1 as a potential therapeutic for dysferlinopathies

Ishemia-Reperfusion enhances GAPDH nitration in aging skeletal muscle

Aging and skeletal muscle ischemia/reperfusion (I/R) injury leads to decreased contractile force generation that increases severely with age. Our studies show that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein expression is significantly decreased at 3 and 5 days reperfusion in the young mouse muscle and at 1, 3, 5, and 7 days in the aged muscle. Using PCR, we have shown that GAPDH mRNA levels in young and old muscle increase at 5 days reperfusion compared to control, suggesting that the protein deficit is not transcriptional. Furthermore, while total tyrosine nitration did not increase in the young muscle, GAPDH nitration increased significantly at 1 and 3 days reperfusion. In contrast, total tyrosine nitration in aged muscle increased significantly at 1, 3, and 5 days of reperfusion, with increases in GAPDH nitration at the same time points. We conclude that GAPDH protein levels decrease following I/R, that this is not transcriptionally mediated, that the aged muscle experiences greater oxidative stress, protein modification and GAPDH degradation, possibly contributing to decreased muscle function. We propose that tyrosine nitration enhances GAPDH degradation following I/R and that the persistent decrease of GAPDH in aged muscle is due to the prolonged increase in oxidative modification in this age group

Resistance of Renal Cell Carcinoma to Sorafenib Is Mediated by Potentially Reversible Gene Expression

Purpose: Resistance to antiangiogenic therapy is an important clinical problem. We examined whether resistance occurs at least in part via reversible, physiologic changes in the tumor, or results solely from stable genetic changes in resistant tumor cells. Experimental Design: Mice bearing two human RCC xenografts were treated with sorafenib until they acquired resistance. Resistant 786-O cells were harvested and reimplanted into naïve mice. Mice bearing resistant A498 cells were subjected to a 1 week treatment break. Sorafenib was then again administered to both sets of mice. Tumor growth patterns, gene expression, viability, blood vessel density, and perfusion were serially assessed in treated vs control mice. Results: Despite prior resistance, reimplanted 786-O tumors maintained their ability to stabilize on sorafenib in sequential reimplantation steps. A transcriptome profile of the tumors revealed that the gene expression profile of tumors upon reimplantation reapproximated that of the untreated tumors and was distinct from tumors exhibiting resistance to sorafenib. In A498 tumors, revascularization was noted with resistance and cessation of sorafenib therapy and tumor perfusion was reduced and tumor cell necrosis enhanced with re-exposure to sorafenib. Conclusions: In two RCC cell lines, resistance to sorafenib appears to be reversible. These results support the hypothesis that resistance to VEGF pathway therapy is not solely the result of a permanent genetic change in the tumor or selection of resistant clones, but rather is due to a great extent to reversible changes that likely occur in the tumor and/or its microenvironment

Changes in symptomatology, reinfection, and transmissibility associated with the SARS-CoV-2 variant B.1.1.7: an ecological study

Background The SARS-CoV-2 variant B.1.1.7 was first identified in December, 2020, in England. We aimed to investigate whether increases in the proportion of infections with this variant are associated with differences in symptoms or disease course, reinfection rates, or transmissibility. Methods We did an ecological study to examine the association between the regional proportion of infections with the SARS-CoV-2 B.1.1.7 variant and reported symptoms, disease course, rates of reinfection, and transmissibility. Data on types and duration of symptoms were obtained from longitudinal reports from users of the COVID Symptom Study app who reported a positive test for COVID-19 between Sept 28 and Dec 27, 2020 (during which the prevalence of B.1.1.7 increased most notably in parts of the UK). From this dataset, we also estimated the frequency of possible reinfection, defined as the presence of two reported positive tests separated by more than 90 days with a period of reporting no symptoms for more than 7 days before the second positive test. The proportion of SARS-CoV-2 infections with the B.1.1.7 variant across the UK was estimated with use of genomic data from the COVID-19 Genomics UK Consortium and data from Public Health England on spike-gene target failure (a non-specific indicator of the B.1.1.7 variant) in community cases in England. We used linear regression to examine the association between reported symptoms and proportion of B.1.1.7. We assessed the Spearman correlation between the proportion of B.1.1.7 cases and number of reinfections over time, and between the number of positive tests and reinfections. We estimated incidence for B.1.1.7 and previous variants, and compared the effective reproduction number, Rt, for the two incidence estimates. Findings From Sept 28 to Dec 27, 2020, positive COVID-19 tests were reported by 36 920 COVID Symptom Study app users whose region was known and who reported as healthy on app sign-up. We found no changes in reported symptoms or disease duration associated with B.1.1.7. For the same period, possible reinfections were identified in 249 (0·7% [95% CI 0·6–0·8]) of 36 509 app users who reported a positive swab test before Oct 1, 2020, but there was no evidence that the frequency of reinfections was higher for the B.1.1.7 variant than for pre-existing variants. Reinfection occurrences were more positively correlated with the overall regional rise in cases (Spearman correlation 0·56–0·69 for South East, London, and East of England) than with the regional increase in the proportion of infections with the B.1.1.7 variant (Spearman correlation 0·38–0·56 in the same regions), suggesting B.1.1.7 does not substantially alter the risk of reinfection. We found a multiplicative increase in the Rt of B.1.1.7 by a factor of 1·35 (95% CI 1·02–1·69) relative to pre-existing variants. However, Rt fell below 1 during regional and national lockdowns, even in regions with high proportions of infections with the B.1.1.7 variant. Interpretation The lack of change in symptoms identified in this study indicates that existing testing and surveillance infrastructure do not need to change specifically for the B.1.1.7 variant. In addition, given that there was no apparent increase in the reinfection rate, vaccines are likely to remain effective against the B.1.1.7 variant. Funding Zoe Global, Department of Health (UK), Wellcome Trust, Engineering and Physical Sciences Research Council (UK), National Institute for Health Research (UK), Medical Research Council (UK), Alzheimer's Society

Activin Receptor Type IIB Inhibition Improves Muscle Phenotype and Function in a Mouse Model of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a devastating neurodegenerative disorder that causes progressive muscle atrophy and weakness. Using adeno-associated virus-mediated gene transfer, we evaluated the potential to improve skeletal muscle weakness via systemic, postnatal inhibition of either myostatin or all signaling via the activin receptor type IIB (ActRIIB). After demonstrating elevated p-SMAD3 content and differential content of ActRIIB ligands, 4-week-old male C/C SMA model mice were treated intraperitoneally with 1x1012 genome copies of pseudotype 2/8 virus encoding a soluble form of the ActRIIB extracellular domain (sActRIIB) or protease-resistant myostatin propeptide (dnMstn) driven by a liver specific promoter. At 12 weeks of age, muscle mass and function were improved in treated C/C mice by both treatments, compared to controls. The fast fiber type muscles had a greater response to treatment than did slow muscles, and the greatest therapeutic effects were found with sActRIIB treatment. Myostatin/activin inhibition, however, did not rescue C/C mice from the reduction in motor unit numbers of the tibialis anterior muscle. Collectively, this study indicates that myostatin/activin inhibition represents a potential therapeutic strategy to increase muscle mass and strength, but not neuromuscular junction defects, in less severe forms of SMA

Activin Receptor Type IIB Inhibition Improves Muscle Phenotype and Function in a Mouse Model of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a devastating neurodegenerative disorder that causes progressive muscle atrophy and weakness. Using adeno-associated virus-mediated gene transfer, we evaluated the potential to improve skeletal muscle weakness via systemic, postnatal inhibition of either myostatin or all signaling via the activin receptor type IIB (ActRIIB). After demonstrating elevated p-SMAD3 content and differential content of ActRIIB ligands, 4-week-old male C/C SMA model mice were treated intraperitoneally with 1x1012 genome copies of pseudotype 2/8 virus encoding a soluble form of the ActRIIB extracellular domain (sActRIIB) or protease-resistant myostatin propeptide (dnMstn) driven by a liver specific promoter. At 12 weeks of age, muscle mass and function were improved in treated C/C mice by both treatments, compared to controls. The fast fiber type muscles had a greater response to treatment than did slow muscles, and the greatest therapeutic effects were found with sActRIIB treatment. Myostatin/activin inhibition, however, did not rescue C/C mice from the reduction in motor unit numbers of the tibialis anterior muscle. Collectively, this study indicates that myostatin/activin inhibition represents a potential therapeutic strategy to increase muscle mass and strength, but not neuromuscular junction defects, in less severe forms of SMA

Increased collagen cross-linking is a signature of dystrophin-deficient muscle.

IntroductionCollagen cross-linking is a key parameter in extracellular matrix (ECM) maturation, turnover, and stiffness. We examined aspects of collagen cross-linking in dystrophin-deficient murine, canine, and human skeletal muscle.MethodsDMD patient biopsies and samples from mdx mice and golden retriever muscular dystrophy dog samples (with appropriate controls) were analyzed. Collagen cross-linking was evaluated using solubility and hydroxyproline assays. Expression of the cross-linking enzyme lysyl oxidase (LOX) was determined by real-time polymerase chain reaction, immunoblotting, and immunofluorescence.ResultsLOX protein levels are increased in dystrophic muscle from all species evaluated. Dystrophic mice and dogs had significantly higher cross-linked collagen than controls, especially in the diaphragm. Distribution of intramuscular LOX was heterogeneous in all samples, but it increased in frequency and intensity in dystrophic muscle.ConclusionThese findings implicate elevated collagen cross-linking as an important component of the disrupted ECM in dystrophic muscles, and heightened cross-linking is evident in mouse, dog, and man. Muscle Nerve 54: 71-78, 2016

Hyperactivation of activin signaling pathway in young SMA^C/C mouse skeletal muscle.

<p>(<b>A-B</b>) Quadriceps muscles from 4 week-old SMA^c/c mice (n = 3) and wild-type (WT) littermates (n = 4) were immunoblotted for phosphorylated and total SMAD3, SMAD1/5/8, and Akt content. (<b>C-D</b>) Protein content of full-length (FL) myostatin (Mstn), FL GDF11, Activin A, and activin IIB receptor (ActRIIB) was measured by immunoblotting IgG-depleted quadriceps lystate samples. Immunoblotting quantifications are normalized to Ponceau Red-visualized loading. Adult muscle and Mstn knockout (KO) samples are included to demonstrate specificity of the antibody used. (<b>E</b>) Relative gene expression of <i>Mstn</i>, <i>Gdf11</i>, <i>Inhba</i>, and <i>Cdkn1a</i> (normalized to <i>Gapdh</i>) of the 4 week-old mouse quadriceps, as measured by real-time PCR. Data were analyzed using Student’s T-tests, and are presented as mean ± SEM with p-values and effect size (<i>d</i>) displayed.</p