Dusp6 is a genetic modifier of growth through enhanced ERK activity.

Like other single-gene disorders, muscular dystrophy displays a range of phenotypic heterogeneity even with the same primary mutation. Identifying genetic modifiers capable of altering the course of muscular dystrophy is one approach to deciphering gene-gene interactions that can be exploited for therapy development. To this end, we used an intercross strategy in mice to map modifiers of muscular dystrophy. We interrogated genes of interest in an interval on mouse chromosome 10 associated with body mass in muscular dystrophy as skeletal muscle contributes significantly to total body mass. Using whole-genome sequencing of the two parental mouse strains combined with deep RNA sequencing, we identified the Met62Ile substitution in the dual-specificity phosphatase 6 (Dusp6) gene from the DBA/2 J (D2) mouse strain. DUSP6 is a broadly expressed dual-specificity phosphatase protein, which binds and dephosphorylates extracellular-signal-regulated kinase (ERK), leading to decreased ERK activity. We found that the Met62Ile substitution reduced the interaction between DUSP6 and ERK resulting in increased ERK phosphorylation and ERK activity. In dystrophic muscle, DUSP6 Met62Ile is strongly upregulated to counteract its reduced activity. We found that myoblasts from the D2 background were insensitive to a specific small molecule inhibitor of DUSP6, while myoblasts expressing the canonical DUSP6 displayed enhanced proliferation after exposure to DUSP6 inhibition. These data identify DUSP6 as an important regulator of ERK activity in the setting of muscle growth and muscular dystrophy.

Clinical features as predictors of histologically confirmed inflammation in patients with lumbar disc herniation with associated radiculopathy.

BACKGROUND: An understanding of the clinical features of inflammation in low back pain with or without leg symptoms may allow targeted evaluations of anti-inflammatory treatment in randomised-controlled-trials and clinical practice. PURPOSE: This study evaluated the diagnostic accuracy of clinical features to predict the presence/absence of histologically confirmed inflammation in herniated disc specimens removed at surgery in patients with lumbar disc herniation and associated radiculopathy (DHR). STUDY DESIGN: Cohort Study. METHODS: Disc material from patients with DHR undergoing lumbar discectomy was sampled and underwent histological/immunohistochemistry analyses. Control discs were sampled from patients undergoing surgical correction for scoliosis. Baseline assessment comprising sociodemographic factors, subjective examination, physical examination and psychosocial screening was conducted and a range of potential clinical predictors of inflammation developed based on the existing literature. Multi-variate analysis was undertaken to determine diagnostic accuracy. RESULTS: Forty patients with DHR and three control patients were recruited. None of the control discs had evidence of inflammation compared to 28% of patients with DHR. Predictors of the presence of histologically confirmed inflammation included back pain < 5/10, symptoms worse the next day after injury, lumbar flexion range between 0 and 30° and a positive clinical inflammation score (at least 3 of: constant symptoms, morning pain/stiffness greater than 60-min, short walking not easing symptoms and significant night symptoms). The model achieved a sensitivity of 90.9%, a specificity of 92.9%, and a predictive accuracy of 92.3%. CONCLUSION: In a sample of patients with lumbar DHR a combination of clinical features predicted the presence or absence of histologically confirmed inflammation. CLINICAL RELEVANCE: These clinical features may enable targeted anti-inflammatory treatment in future RCTs and in clinical practice.

A multivariate prognostic model for pain and activity limitation in people undergoing lumbar discectomy.

Purpose: The purpose of this study was to identify a multivariate predictive model for 6-month outcomes on overall pain, leg pain and activity limitation in patients undergoing lumbar discectomy. Identification of predictors of outcome for lumbar discectomy has the potential to assist identifying treatment targets, clinical decision making and disease understanding.Materials and methods: Prospective cohort design. Ninety-seven patients deemed by study surgeons to be suitable for lumbar discectomy completed a comprehensive clinical and radiological baseline assessment. At 6-months post surgery outcome measures of overall and leg pain (visual analogue scale) as well as activity limitation (Oswestry Disability Index) were completed. Univariate and multivariate analyses were conducted to determine the best multivariate predictive model of outcome.Results: In the multivariate model, presence of a compensation claim, longer duration of injury and presence of below knee pain and/or parasthesia were negative prognostic indicators for at least two of the outcomes. Peripheralization in response to mechanical loading strategies was a positive prognostic indicator for overall pain and leg pain. A range of other prognostic indicators for one outcome were also identified. The prognostic model explained up to 32% of the variance in outcome.Conclusions: An 11-factor prognostic model was identified from a range of clinically and radiologically assessed variables in accordance with a biopsychosocial model. The multivariate model has potential implications for researchers and practitioners in the field. Further high quality research is required to externally validate the prognostic model, evaluate effect of the identified prognostic factors on treatment effectiveness and explore potential mechanisms of effect.

Intermittent Glucocorticoid Dosing Improves Muscle Repair and Function in Mice with Limb-Girdle Muscular Dystrophy.

The muscular dystrophies are genetically diverse. Shared pathological features among muscular dystrophies include breakdown, or loss of muscle, and accompanying fibrotic replacement. Novel strategies are needed to enhance muscle repair and function and to slow this pathological remodeling. Glucocorticoid steroids, like prednisone, are known to delay loss of ambulation in patients with Duchenne muscular dystrophy but are accompanied by prominent adverse effects. However, less is known about the effects of steroid administration in other types of muscular dystrophies, including limb-girdle muscular dystrophies (LGMDs). LGMD 2B is caused by loss of dysferlin, a membrane repair protein, and LGMD 2C is caused by loss of the dystrophin-associated protein, γ-sarcoglycan. Herein, we assessed the efficacy of steroid dosing on sarcolemmal repair, muscle function, histopathology, and the regenerative capacity of primary muscle cells. We found that in murine models of LGMD 2B and 2C, daily prednisone dosing reduced muscle damage and fibroinflammatory infiltration. However, daily prednisone dosing also correlated with increased muscle adipogenesis and atrophic remodeling. Conversely, intermittent dosing of prednisone, provided once weekly, enhanced muscle repair and did not induce atrophy or adipogenesis, and was associated with improved muscle function. These data indicate that dosing frequency of glucocorticoid steroids affects muscle remodeling in non-Duchenne muscular dystrophies, suggesting a positive outcome associated with intermittent steroid dosing in LGMD 2B and 2C muscle.

Sarcoidosis following alemtuzumab treatment for multiple sclerosis.

Despite proven efficacy of alemtuzumab in multiple sclerosis (MS), approximately 50% of individuals will develop a new autoimmune disease following treatment. To date, these have largely been antibody mediated and organ specific (primarily affecting the thyroid gland). In a retrospective case series of 187 patients from two UK specialist centres (Cardiff and Cambridge) followed up for a median of 10 years, we report three (1.6%) cases of sarcoidosis following alemtuzumab treatment of MS. This report increases the spectrum of auto-inflammatory disease following alemtuzumab and should be considered by clinicians when using this therapeutic agent for MS.

The effect of alphacypermethrin-treated mesh protection against African horse sickness virus vectors on jet stall microclimate, clinical variables and faecal glucocorticoid metabolites of horses.

BACKGROUND: African horse sickness (AHS) is of importance to health and international trade in horses worldwide. During export from and transit through AHS endemic countries or zones, physical and chemical measures to protect horses from the vectors of AHS virus (AHSV) are recommended by the World Organization for Animal Health. Protection of containerized air transport systems for horses (jet stalls) with alphacypermethrin insecticide-treated high density polyethylene mesh is effective in reducing the Culicoides midge vector attack rate. In order to determine the effect of this mesh on jet stall ventilation and horse welfare under temperate climatic conditions, jet stall microclimate, clinical variables and faecal glucocorticoid metabolite (FGM) levels of 12 horses were monitored during overnight housing in either a treated or untreated stall in two blocks of a 2 × 3 randomized crossover design. RESULTS: Temperature difference between the treated stall and outside was significantly higher than the difference between the untreated stall and outside at 1/15 time points only (P = 0.045, r = 0.70). Relative humidity (RH) difference between the treated stall and outside did not differ from the untreated stall and outside. Temperature and RH in the treated stall were highly and significantly correlated with outside temperature (r = 0.96, P < 0.001) and RH (r = 0.95, P < 0.001), respectively. No significant differences were detected between rectal temperatures, pulse and respiratory rates of horses in the treated stall compared to the untreated stall. Mean FGM concentrations for horses housed in the treated stall peaked earlier (24 h) and at a higher concentration than horses housed in the untreated stall (48 h), but were not significantly different from baseline. No significant difference was detected in FGM concentrations when the treated and untreated stall groups were compared at individual time points up to 72 h after exiting the jet stall. CONCLUSIONS: Alphacypermethrin-treated HDPE mesh could be used under temperate climatic conditions to protect horses in jet stalls against AHSV vectors, without compromising jet stall microclimate and horse welfare.

Isolation and molecular characterization of Mycobacterium bovis causing pulmonary tuberculosis and epistaxis in a Thoroughbred horse.

BACKGROUND: Tuberculosis caused by Mycobacterium bovis (M. bovis) is very uncommon in horses worldwide. CASE PRESENTATION: In the current study, an eight-year-old male Thoroughbred in good body condition was admitted to the Equine Clinic at the Onderstepoort Veterinary Academic Hospital in 2005 due to bilateral epistaxis accompanied by coughing. Routine examinations were conducted to determine the cause of the condition. Endoscopic examination revealed the major source of the epistaxis as the trachea, whereas thoracic radiography indicated the presence of a primary pulmonary mass. M. bovis was isolated from a broncho-alveolar lavage (BAL) sample collected. The pulmonary mass reduced in size three months later following an oral administration of enrofloxacin (7.5 mg/kg PO SID). Genetic fingerprinting by spoligotyping identified the M. bovis isolate as spoligotype SB0868 strain. This M. bovis strain type was never described previously in South Africa (SA). This is the first case of M. bovis infection in a horse in SA which has been fully documented including clinical findings, isolation and genetic characterisation of the causative pathogen. CONCLUSIONS: This report indicates that horses may contract and harbour M. bovis despite their lower susceptibility compared to other domestic animals. It also suggests that the infection may be more easily contained and eliminated from the host.

Detection of equine herpesvirus-4 and physiological stress patterns in young Thoroughbreds consigned to a South African auction sale.

BACKGROUND: The prevalence of equine herpesvirus types-1 and -4 (EHV-1 and -4) in South African Thoroughbreds at auction sales is currently undefined. Commingling of young Thoroughbreds from various populations together with physiological stress related to their transport and confinement at a sales complex, may be associated with shedding and transmission of EHV-1 and -4. This prospective cohort study sampled 90 young Thoroughbreds consigned from eight farms, originating from three provinces representative of the South African Thoroughbred breeding demographic to a sales complex. Nasal swabs for quantitative real-time polymerase chain reaction (qPCR) assay to detect EHV-1 and -4 nucleic acid and blood samples for enzyme-linked immunosorbent assay for EHV-1 and -4 antibodies were collected from all horses on arrival and departure. Additional nasal swabs for qPCR were obtained serially from those displaying pyrexia and, or nasal discharge. Daily faecal samples were used for determination of faecal glucocorticoid metabolite (FGM) concentrations as a measurement of physiological stress and these values were modelled to determine the factors best explaining FGM variability. RESULTS: EHV-4 nucleic acid was detected in 14.4 % and EHV-1 from none of the animals in the study population. Most (93.3 %) and very few (1.1 %) of this population showed antibodies indicating prior exposure to EHV-4 and EHV-1 respectively. Pyrexia and nasal discharge were poor predictors for detecting EHV-4 nucleic acid. The horses' FGM concentrations increased following arrival before decreasing for most of the remaining study period including the auction process. Model averaging showed that variation in FGM concentrations was best explained by days post-arrival and transport duration. CONCLUSIONS: In this study population, sales consignment was associated with limited detection of EHV-4 nucleic acid in nasal secretions, with most showing prior exposure to EHV-4 and very few to EHV-1. The physiological stress response shown by most reflected the combination of stressors associated with transport and arrival and these are key areas for future investigation into management practices to enhance health and welfare of young Thoroughbreds during sales consignment.

Effects of xylazine, romifidine, or detomidine on hematology, biochemistry, and splenic thickness in healthy horses.

Alpha-2 agonist-induced changes in packed cell volume (PCV), total solids (TS), selected biochemical parameters, and splenic thickness were investigated in horses. Four healthy mares were treated in a blinded, randomized, cross-over design with a dose of xylazine (0.5 mg/kg), romifidine (0.04 mg/kg), or detomidine (0.01 mg/kg) IV, and detomidine (0.02 mg/kg) IM. Hematology, TS, colloid osmotic pressure (COP), plasma osmolality; glucose, lactate, urea (BUN) and electrolyte concentrations; venous blood pH and ultrasonographic splenic thickness were evaluated at intervals for 300 min. Repeated measures analysis of variance (ANOVA) were performed with P < 0.05. There was a significant change over time in PCV and TS following each treatment (P < 0.001), with median (range) reductions of 20.9% (12.9% to 27.3%) and 5.8% (3.0% to 10.3%), respectively. Red blood cell count, BUN, and COP decreased while osmolality, glucose, Na(+), and splenic thickness increased. Treatments induced clinically significant transient changes in PCV, TS, and other biochemical parameters, which should be considered when assessing horses that received these drugs.

Effets de la xylazine, de la romifidine ou de la détomidine sur l'hématologie, la biochimie et l'épaisseur splénique chez des chevaux en santé. Des changements induits à l'aide d'alpha-2 agonistes au niveau de la valeur d'hématocrite (VH), des solides totaux (ST), de paramètres biochimiques choisis et de l'épaisseur splénique ont été étudiés chez les chevaux. Quatre juments en santé ont été traitées dans une étude à l'aveugle, randomisée et croisée avec une dose de xylazine (0,5 mg/kg), de romifidine (0,04 mg/kg) ou de détomidine (0,01 mg/kg) IV et de détomidine (0,02 mg/kg) IM. L'hématologie, les ST, la pression osmotique colloïdale (POC), l'osmolalité plasmatique, le glucose, le lactate, l'urée (l'azote uréique du sang) et les concentrations d'électrolytes, le pH du sang veineux et l'épaisseur splénique échographique ont été évalués à des intervalles de 300 minutes. L'analyse de la variance des mesures répétées a été réalisée avec P < 0,05. Il y avait des changements significatifs dans le temps de VH et des ST après chaque traitement (P < 0,001), avec des réductions moyennes (écart) de 20,9 % (de 12,9 % à 27,3 %) et de 5,8 % (de 3,0 % à 10,3 %), respectivement. Le nombre de globules rouges, l'azote uréique du sang et la POC ont diminué tandis que l'osmolalité, le glucose, Na+ et l'épaisseur splénique ont augmenté. Les traitements ont induit des changements transitoires significatifs sur le plan clinique dans la VH, les ST et les autres paramètres biochimiques qui devraient être considérés lors de l'évaluation des chevaux qui ont reçu ces médicaments.(Traduit par Isabelle Vallières).

The extracellular matrix differentially directs myoblast motility and differentiation in distinct forms of muscular dystrophy: Dystrophic matrices alter myoblast motility.

Extracellular matrix (ECM) pathologic remodeling underlies many disorders, including muscular dystrophy. Tissue decellularization removes cellular components while leaving behind ECM components. We generated "on-slide" decellularized tissue slices from genetically distinct dystrophic mouse models. The ECM of dystrophin- and sarcoglycan-deficient muscles had marked thrombospondin 4 deposition, while dysferlin-deficient muscle had excess decorin. Annexins A2 and A6 were present on all dystrophic decellularized ECMs, but annexin matrix deposition was excessive in dysferlin-deficient muscular dystrophy. Muscle-directed viral expression of annexin A6 resulted in annexin A6 in the ECM. C2C12 myoblasts seeded onto decellularized matrices displayed differential myoblast mobility and fusion. Dystrophin-deficient decellularized matrices inhibited myoblast mobility, while dysferlin-deficient decellularized matrices enhanced myoblast movement and differentiation. Myoblasts treated with recombinant annexin A6 increased mobility and fusion like that seen on dysferlin-deficient decellularized matrix and demonstrated upregulation of ECM and muscle cell differentiation genes. These findings demonstrate specific fibrotic signatures elicit effects on myoblast activity.

The super-healing MRL strain promotes muscle growth in muscular dystrophy through a regenerative extracellular matrix.

The Murphy Roths Large (MRL) mouse strain has "super-healing" properties that enhance recovery from injury. In mice, the DBA/2J strain intensifies many aspects of muscular dystrophy, so we evaluated the ability of the MRL strain to suppress muscular dystrophy in the Sgcg-null mouse model of limb girdle muscular dystrophy. A comparative analysis of Sgcg-null mice in the DBA/2J versus MRL strains showed greater myofiber regeneration, with reduced structural degradation of muscle in the MRL strain. Transcriptomic profiling of dystrophic muscle indicated strain-dependent expression of extracellular matrix (ECM) and TGF-ß signaling genes. To investigate the MRL ECM, cellular components were removed from dystrophic muscle sections to generate decellularized myoscaffolds. Decellularized myoscaffolds from dystrophic mice in the protective MRL strain had significantly less deposition of collagen and matrix-bound TGF-ß1 and TGF-ß3 throughout the matrix. Dystrophic myoscaffolds from the MRL background, but not the DBA/2J background, were enriched in myokines like IGF-1 and IL-6. C2C12 myoblasts seeded onto decellularized matrices from Sgcg-/- MRL and Sgcg-/- DBA/2J muscles showed the MRL background induced greater myoblast differentiation compared with dystrophic DBA/2J myoscaffolds. Thus, the MRL background imparts its effect through a highly regenerative ECM, which is active even in muscular dystrophy.

Nicotinamide adenine dinucleotide phosphate oxidase in experimental liver fibrosis: GKT137831 as a novel potential therapeutic agent.

UNLABELLED: Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) generates reactive oxygen species (ROS) in hepatic stellate cells (HSCs) during liver fibrosis. In response to fibrogenic agonists, such as angiotensin II (Ang II), the NOX1 components form an active complex, including Ras-related botulinum toxin substrate 1 (Rac1). Superoxide dismutase 1 (SOD1) interacts with the NOX-Rac1 complex to stimulate NOX activity. NOX4 is also induced in activated HSCs/myofibroblast by increased gene expression. Here, we investigate the role of an enhanced activity SOD1 G37R mutation (SODmu) and the effects of GKT137831, a dual NOX1/4 inhibitor, on HSCs and liver fibrosis. To induce liver fibrosis, wild-type (WT) and SOD1mu mice were treated with CCl(4) or bile duct ligation (BDL). Then, to address the role of NOX-SOD1-mediated ROS production in HSC activation and liver fibrosis, mice were treated with a NOX1/4 inhibitor. Fibrosis and ROS generation was assessed by histology and measurement of thiobarbituric acid reactive substances and NOX-related genes. Primary cultured HSCs isolated from WT, SODmu, and NOX1 knockout (KO) mice were assessed for ROS production, Rac1 activity, and NOX gene expression. Liver fibrosis was increased in SOD1mu mice, and ROS production and Rac1 activity were increased in SOD1mu HSCs. The NOX1/4 inhibitor, GKT137831, attenuated liver fibrosis and ROS production in both SOD1mu and WT mice as well as messenger RNA expression of fibrotic and NOX genes. Treatment with GKT137831 suppressed ROS production and NOX and fibrotic gene expression, but not Rac1 activity, in SOD1mut and WT HSCs. Both Ang II and tumor growth factor beta up-regulated NOX4, but Ang II required NOX1. CONCLUSIONS: SOD1mu induces excessive NOX1 activation through Rac1 in HSCs, causing enhanced NOX4 up-regulation, ROS generation, and liver fibrosis. Treatment targeting NOX1/4 may be a new therapy for liver fibrosis.

Renoprotective effects of a novel Nox1/4 inhibitor in a mouse model of Type 2 diabetes.

Nox (NADPH oxidase)-derived ROS (reactive oxygen species) have been implicated in the development of diabetic nephropathy. Of the Nox isoforms in the kidney, Nox4 is important because of its renal abundance. In the present study, we tested the hypothesis that GKT136901, a Nox1/4 inhibitor, prevents the development of nephropathy in db/db (diabetic) mice. Six groups of male mice (8-week-old) were studied: (i) untreated control db/m, (ii) low-dose GKT136901-treated db/m (30 mg/kg of body weight per day), (iii) high-dose GKT136901-treated db/m (90 mg/kg of body weight per day), (iv) untreated db/db; (v) low dose GKT136901-treated db/db; and (vi) high-dose GKT136901-treated db/db. GKT136901, in chow, was administered for 16 weeks. db/db mice developed diabetes and nephropathy as evidenced by hyperglycaemia, albuminuria and renal injury (mesangial expansion, tubular dystrophy and glomerulosclerosis). GKT136901 treatment had no effect on plasma glucose or BP (blood pressure) in any of the groups. Plasma and urine TBARSs (thiobarbituric acid-reacting substances) levels, markers of systemic and renal oxidative stress, respectively, were increased in diabetic mice. Renal mRNA expression of Nox4, but not of Nox2, increased, Nox1 was barely detectable in db/db. Expression of the antioxidant enzyme SOD-1 (superoxide dismutase 1) decreased in db/db mice. Renal content of fibronectin, pro-collagen, TGFß (transforming growth factor ß) and VCAM-1 (vascular cell adhesion molecule 1) and phosphorylation of ERK1/2 (extracellular-signal-regulated kinase 1/2) were augmented in db/db kidneys, with no change in p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase). Treatment reduced albuminuria, TBARS and renal ERK1/2 phosphorylation and preserved renal structure in diabetic mice. Our findings suggest a renoprotective effect of the Nox1/4 inhibitor, possibly through reduced oxidative damage and decreased ERK1/2 activation. These phenomena occur independently of improved glucose control, suggesting GKT136901-sensitive targets are involved in complications of diabetes rather than in the disease process.

The super-healing MRL strain promotes muscle growth in muscular dystrophy through a regenerative extracellular matrix.

Genetic background shifts the severity of muscular dystrophy. In mice, the DBA/2J strain confers a more severe muscular dystrophy phenotype, whereas the Murphy's Roth Large (MRL) strain has "super-healing" properties that reduce fibrosis. A comparative analysis of the Sgcg null model of Limb Girdle Muscular Dystrophy in the DBA/2J versus MRL strain showed the MRL background was associated with greater myofiber regeneration and reduced structural degradation of muscle. Transcriptomic profiling of dystrophic muscle in the DBA/2J and MRL strains indicated strain-dependent expression of the extracellular matrix (ECM) and TGF-ß signaling genes. To investigate the MRL ECM, cellular components were removed from dystrophic muscle sections to generate decellularized "myoscaffolds". Decellularized myoscaffolds from dystrophic mice in the protective MRL strain had significantly less deposition of collagen and matrix-bound TGF-ß1 and TGF-ß3 throughout the matrix, and dystrophic myoscaffolds from the MRL background were enriched in myokines. C2C12 myoblasts were seeded onto decellularized matrices from Sgcg-/- MRL and Sgcg-/- DBA/2J matrices. Acellular myoscaffolds from the dystrophic MRL background induced myoblast differentiation and growth compared to dystrophic myoscaffolds from the DBA/2J matrices. These studies establish that the MRL background also generates its effect through a highly regenerative ECM, which is active even in muscular dystrophy.

The Nox4 inhibitor GKT137831 attenuates hypoxia-induced pulmonary vascular cell proliferation.

Increased NADP reduced (NADPH) oxidase 4 (Nox4) and reduced expression of the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) contribute to hypoxia-induced pulmonary hypertension (PH). To examine the role of Nox4 activity in pulmonary vascular cell proliferation and PH, the current study used a novel Nox4 inhibitor, GKT137831, in hypoxia-exposed human pulmonary artery endothelial or smooth muscle cells (HPAECs or HPASMCs) in vitro and in hypoxia-treated mice in vivo. HPAECs or HPASMCs were exposed to normoxia or hypoxia (1% O(2)) for 72 hours with or without GKT137831. Cell proliferation and Nox4, PPARγ, and transforming growth factor (TGF)ß1 expression were measured. C57Bl/6 mice were exposed to normoxia or hypoxia (10% O(2)) for 3 weeks with or without GKT137831 treatment during the final 10 days of exposure. Lung PPARγ and TGF-ß1 expression, right ventricular hypertrophy (RVH), right ventricular systolic pressure (RVSP), and pulmonary vascular remodeling were measured. GKT137831 attenuated hypoxia-induced H(2)O(2) release, proliferation, and TGF-ß1 expression and blunted reductions in PPARγ in HPAECs and HPASMCs in vitro. In vivo GKT137831 inhibited hypoxia-induced increases in TGF-ß1 and reductions in PPARγ expression and attenuated RVH and pulmonary artery wall thickness but not increases in RVSP or muscularization of small arterioles. This study shows that Nox4 plays a critical role in modulating proliferative responses of pulmonary vascular wall cells. Targeting Nox4 with GKT137831 provides a novel strategy to attenuate hypoxia-induced alterations in pulmonary vascular wall cells that contribute to vascular remodeling and RVH, key features involved in PH pathogenesis.

Intermittent glucocorticoid treatment enhances skeletal muscle performance through sexually dimorphic mechanisms.

Glucocorticoid steroids are commonly prescribed for many inflammatory conditions, but chronic daily use produces adverse effects, including muscle wasting and weakness. In contrast, shorter glucocorticoid pulses may improve athletic performance, although the mechanisms remain unclear. Muscle is sexually dimorphic and comparatively little is known about how male and female muscles respond to glucocorticoids. We investigated the impact of once-weekly glucocorticoid exposure on skeletal muscle performance comparing male and female mice. One month of once-weekly glucocorticoid dosing improved muscle specific force in both males and females. Transcriptomic profiling of isolated myofibers identified a striking sexually dimorphic response to weekly glucocorticoids. Male myofibers had increased expression of genes in the IGF1/PI3K pathway and calcium handling, while female myofibers had profound upregulation of lipid metabolism genes. Muscles from weekly prednisone-treated males had improved calcium handling, while comparably treated female muscles had reduced intramuscular triglycerides. Consistent with altered lipid metabolism, weekly prednisone-treated female mice had greater endurance relative to controls. Using chromatin immunoprecipitation, we defined a sexually dimorphic chromatin landscape after weekly prednisone. These results demonstrate that weekly glucocorticoid exposure elicits distinct pathways in males versus females, resulting in enhanced performance.

A conserved annexin A6-mediated membrane repair mechanism in muscle, heart, and nerve.

Membrane instability and disruption underlie myriad acute and chronic disorders. Anxa6 encodes the membrane-associated protein annexin A6 and was identified as a genetic modifier of muscle repair and muscular dystrophy. To evaluate annexin A6's role in membrane repair in vivo, we inserted sequences encoding green fluorescent protein (GFP) into the last coding exon of Anxa6. Heterozygous Anxa6gfp mice expressed a normal pattern of annexin A6 with reduced annexin A6GFP mRNA and protein. High-resolution imaging of wounded muscle fibers showed annexin A6GFP rapidly formed a repair cap at the site of injury. Injured cardiomyocytes and neurons also displayed repair caps after wounding, highlighting annexin A6-mediated repair caps as a feature in multiple cell types. Using surface plasmon resonance, we showed recombinant annexin A6 bound phosphatidylserine-containing lipids in a Ca2+- and dose-dependent fashion with appreciable binding at approximately 50 µM Ca2+. Exogenously added recombinant annexin A6 localized to repair caps and improved muscle membrane repair capacity in a dose-dependent fashion without disrupting endogenous annexin A6 localization, indicating annexin A6 promotes repair from both intracellular and extracellular compartments. Thus, annexin A6 orchestrates repair in multiple cell types, and recombinant annexin A6 may be useful in additional chronic disorders beyond skeletal muscle myopathies.

NADPH oxidases regulate CD44 and hyaluronic acid expression in thrombin-treated vascular smooth muscle cells and in atherosclerosis.

The intracellular signaling events by which NADPH oxidase-generated reactive oxygen species (ROS) modulate vascular smooth muscle cell (VSMC) function and atherogenesis are yet to be entirely elucidated. We previously demonstrated that NADPH oxidase deficiency decreased atherosclerosis in apoE(-/-) mice and identified adhesion protein CD44 as an important ROS-sensitive gene expressed in VSMC and atherosclerotic lesions. Here, we examined the molecular mechanisms by which NADPH oxidase-generated ROS regulate the expression of CD44 and its principal ligand, hyaluronan (HA), and how CD44-HA interaction affects VSMC proliferation and migration and inflammatory gene expression in apoE(-/-) mice aortas. Thrombin-induced CD44 expression is mediated by transcription factor AP-1 in a NADPH oxidase-dependent manner. NADPH oxidase-mediated ROS generation enhanced thrombin-induced HA synthesis, and hyaluronan synthase 2 expression in VSMC. Hyaluronidase, which generates low molecular weight HA (LMW-HA), is induced in VSMC in a NADPH oxidase-dependent manner and LMW-HA stimulated ROS generation and cell proliferation in wild-type but not p47(phox-/-) VSMC, effects that were enhanced by thrombin pretreatment. Haptotactic VSMC migration toward HA was increased by thrombin in a CD44-dependent manner. HA expression in atherosclerotic lesions and plasma-soluble CD44 and HA levels were higher in apoE(-/-) compared with apoE(-/-)/p47(phox-/-) mice. HA-regulated pro-inflammatory gene expression was higher in apoE(-/-) than apoE(-/-)/p47(phox-/-) mouse aortas. GKT136901, a specific inhibitor of Nox1- and Nox4-containing NADPH oxidase activity, attenuated ROS generation and atherosclerosis and decreased CD44 and HA expression in atherosclerotic lesions. Together, these data suggest that increased CD44 and HA expression and CD44-HA-dependent gene regulation may play a role in atherosclerosis stimulated by NADPH oxidase activation.

Design, synthesis and biological activity of original pyrazolo-pyrido-diazepine, -pyrazine and -oxazine dione derivatives as novel dual Nox4/Nox1 inhibitors.

Pyrazolo-pyrido-diazepine, -pyrazine and -oxazine dione derivatives are new chemical entities with good and attractive druglikeness properties. A series of pyrazolo-pyrido-diazepine dione analogs demonstrated to be particularly amenable to lead optimization through a couple of cycles in order to improve specificity for isoforms Nox4 and Nox1 and had excellent pharmacokinetic parameters by oral route. Several molecules such as compound 7c proved to be highly potent in in vitro assays on human lung fibroblasts differentiation as well as in curative murine models of bleomycin-induced pulmonary fibrosis with superior efficiency over Pirfenidone. Pyrazolo-pyrido-diazepine dione derivatives targeting Nox4 and Nox1 isoforms appear highly promising therapeutics for the treatment of idiopathic pulmonary fibrosis.

Association of Continuous Intraoperative Vasopressor Use With Reoperation Rates in Head and Neck Free-Flap Reconstruction.

Importance: Continuous vasopressor use in free-flap reconstruction is a point of contention among microvascular surgeons despite data demonstrating safety. Objective: To investigate the association between continuous vasopressor use and the incidence of reoperation in the early postoperative period. Design, Setting, and Participants: In this cohort study, a retrospective medical record review was conducted of patients who underwent head and neck free-flap reconstructions between May 1, 2014, and October 31, 2019, in an academic tertiary care center. All patients undergoing free-flap reconstruction for head and neck defects were included. Exposures: Continuous intraoperative vasopressors. Main Outcomes and Measures: Patient medical records were queried for demographic variables; intraoperative use of vasopressors; vasopressor type, duration, and infusion rate; reoperation within the first 5 postoperative days; and reason for reoperation. Results: Four hundred forty-nine consecutive free-flap reconstructions were performed on 426 patients. The mean age was 62 years (IQR, 55.7-71.1); 293 patients were men (65.3%), 380 were White (84.6%), 55 were Black (12.2%), and 14 were of other race or ethnicity (3.1%). A total of 174 patients received a continuous vasopressor during their reconstruction. Twenty-three reoperations occurred within 5 days postoperatively, 8 of which included vasopressors during initial intervention. Vasopressor type had no association with reoperation (4.5% vs 5.5% [8/174 vs 15/275, respectively] for patients who received vasopressors vs those who did not) (dobutamine odds ratio [OR], 1.02 [95% CI, 0.21-2.91]; dopamine OR, 1.48 [95% CI, 0.33-4.26]). No difference was seen in the duration (dobutamine OR, 1.50 [95% CI, 0.78-2.90]; dopamine OR, 0.87 [95% CI, 0.59-1.28]) or infusion rate (dobutamine OR, 1.50 [95% CI, 0.99-1.02]; dopamine OR, 1.00 [95% CI, 0.99-1.01]) of vasopressors between patients who underwent reoperation and those who did not. Analysis after the exclusion of reasons for reoperation that did not represent possible microvascular anastomosis failure (eg, Doppler malfunction, donor site complications) showed no increased propensity for reoperation (OR, 1.18; 95% CI, 0.27-3.9). Conclusions and Relevance: In this cohort study, use of vasopressors for extensive periods intraoperatively during free-tissue transfer appeared to have no association with the rate of reoperation within 5 days of intervention, regardless of agent used, simultaneous use of agents, type of free-flap operation performed, or reason for reoperation. This study adds to the body of literature supporting the judicious use of vasopressors in patients requiring intraoperative pharmacological pressure support during free-flap reconstruction.