Quiescent Ovarian Cancer Cells Secrete Follistatin to Induce Chemotherapy Resistance in Surrounding Cells in Response to Chemotherapy.

PURPOSE: We recently reported that the transcription factor NFATC4, in response to chemotherapy, drives cellular quiescence to increase ovarian cancer chemoresistance. The goal of this work was to better understand the mechanisms of NFATC4-driven ovarian cancer chemoresistance. EXPERIMENTAL DESIGN: We used RNA sequencing to identify NFATC4-mediated differential gene expression. CRISPR-Cas9 and FST (follistatin)-neutralizing antibodies were used to assess impact of loss of FST function on cell proliferation and chemoresistance. ELISA was used to quantify FST induction in patient samples and in vitro in response to chemotherapy. RESULTS: We found that NFATC4 upregulates FST mRNA and protein expression predominantly in quiescent cells and FST is further upregulated following chemotherapy treatment. FST acts in at least a paracrine manner to induce a p-ATF2-dependent quiescent phenotype and chemoresistance in non-quiescent cells. Consistent with this, CRISPR knockout (KO) of FST in ovarian cancer cells or antibody-mediated neutralization of FST sensitizes ovarian cancer cells to chemotherapy treatment. Similarly, CRISPR KO of FST in tumors increased chemotherapy-mediated tumor eradication in an otherwise chemotherapy-resistant tumor model. Suggesting a role for FST in chemoresistance in patients, FST protein in the abdominal fluid of patients with ovarian cancer significantly increases within 24 hours of chemotherapy exposure. FST levels decline to baseline levels in patients no longer receiving chemotherapy with no evidence of disease. Furthermore, elevated FST expression in patient tumors is correlated with poor progression-free, post-progression-free, and overall survival. CONCLUSIONS: FST is a novel therapeutic target to improve ovarian cancer response to chemotherapy and potentially reduce recurrence rates.

Nanoparticle-Based Follistatin Messenger RNA Therapy for Reprogramming Metastatic Ovarian Cancer and Ameliorating Cancer-Associated Cachexia.

This study presents the first messenger RNA (mRNA) therapy for metastatic ovarian cancer and cachexia-induced muscle wasting based on lipid nanoparticles that deliver follistatin (FST) mRNA predominantly to cancer clusters following intraperitoneal administration. The secreted FST protein, endogenously synthesized from delivered mRNA, efficiently reduces elevated activin A levels associated with aggressive ovarian cancer and associated cachexia. By altering the cancer cell phenotype, mRNA treatment prevents malignant ascites, delays cancer progression, induces the formation of solid tumors, and preserves muscle mass in cancer-bearing mice by inhibiting negative regulators of muscle mass. Finally, mRNA therapy provides synergistic effects in combination with cisplatin, increasing the survival of mice and counteracting muscle atrophy induced by chemotherapy and cancer-associated cachexia. The treated mice develop few nonadherent tumors that are easily resected from the peritoneum. Clinically, this nanomedicine-based mRNA therapy can facilitate complete cytoreduction, target resistance, improve resilience during aggressive chemotherapy, and improve survival in advanced ovarian cancer.

Pre-treatment serum protein levels predict survival of non-small cell lung cancer patients without durable clinical benefit by PD-1/L1 inhibitors.

While PD-1/L1 inhibitors are characterized by durable tumor control, they also prolong survival without prolongation of progression-free survival (PFS) in part of patients. However, little is known about the factors and mechanisms involved in this. Between December 2015 and September 2018, 106 patients with advanced non-small cell lung cancer treated with ICI monotherapy were enrolled in a prospective-observational study. Sixty-nine of whom progressed or died within 6 months after ICI initiation were defined as patients without durable clinical benefit (NDBs). Clinical factors and 39 serum proteins before ICI initiation and at the time of progressive disease (PD) were explored for an association with overall survival (OS) and OS after PD (OS-PD). As a result, median PFS, OS, and OS-PD were 44 days [95% confidence interval (CI): 39-56), 211 days (95% CI: 158-425), and 193 days (95% CI: 118-349), respectively. By multivariate analysis for OS, CRP (> 1.44 mg/dl) [HR 2.59 (95% CI:1.33-5.04), P = 0.005] and follistatin (> 685 pg/ml) [HR 2.29 (95% CI:1.12-4.69), P = 0.023] before ICI initiation were significantly predictive. Notably, no serum protein at the time of PD was predictive for OS-PD. There were also no serum predictive factors of OS in the 33 patients with durable clinical benefit. In conclusion, serum levels of CRP and follistatin before ICI initiation, not at the time of PD, are predictive for OS in NDBs, suggesting long-term survivor in NDBs are predetermined by the immune status before ICI initiation.

Activin A triggers angiogenesis via regulation of VEGFA and its overexpression is associated with poor prognosis of oral squamous cell carcinoma.

Poor prognosis associated with the dysregulated expression of activin A in a number of malignancies has been related to with numerous aspects of tumorigenesis, including angiogenesis. The present study investigated the prognostic significance of activin A immunoexpression in blood vessels and cancer cells in a number of oral squamous cell carcinoma (OSCC) cases and applied in vitro strategies to determine the impact of activin A on angiogenesis. In a cohort of 95 patients with OSCC, immunoexpression of activin A in both blood vessels and tumor cells was quantified and the association with clinicopathological parameters and survival was analyzed. Effects of activin A on the tube formation, proliferation and migration of human umbilical vein endothelial cells (HUVECs) were evaluated in gain­of­function (treatment with recombinant activin A) or loss­of­function [treatment with activin A­antagonist follistatin or by stable transfection with short hairpin RNA (shRNA) targeting activin A] conditions. Conditioned medium from an OSCC cell line with shRNA­mediated depletion of activin A was also tested. The profile of pro­ and anti­angiogenic factors regulated by activin A was assessed with a human angiogenesis quantitative PCR (qPCR) array. Vascular endothelial growth factor A (VEGFA) and its major isoforms were evaluated by reverse transcription­qPCR and ELISA. Activin A expression in blood vessels demonstrated an independent prognostic value in the multivariate analysis with a hazard ratio of 2.47 [95% confidence interval (CI), 1.30­4.71; P=0.006) for disease­specific survival and 2.09 (95% CI, 1.07­4.08l: P=0.03) for disease­free survival. Activin A significantly increased tubular formation of HUVECs concomitantly with an increase in proliferation. This effect was validated by reduced proliferation and tubular formation of HUVECs following inhibition of activin A by follistatin or shRNA, as well as by treatment of HUVECs with conditioned medium from activin A­depleted OSCC cells. Activin A­knockdown increased the migration of HUVECs. In addition, activin A stimulated the phosphorylation of SMAD2/3 and the expression and production of total VEGFA, significantly enhancing the expression of its pro­angiogenic isoform 121. The present findings suggest that activin A is a predictor of the prognosis of patients with OSCC, and provide evidence that activin A, in an autocrine and paracrine manner, may contribute to OSCC angiogenesis through differential expression of the isoform 121 of VEGFA.

Exogenous follistatin administration ameliorates cisplatin-induced acute kidney injury through anti-inflammation and anti-apoptosis effects.

OBJECTIVES: This study was aimed to explore the effects of follistatin on cisplatin-induced renal dysfunction, histopathological changes, apoptosis, inflammation and oxidative damage in rats. BACKGROUND: Follistatin plays an important role in the developmental and regeneration processes of kidney by blocking the actions of activin, which is a member of transforming growth factor-ß superfamily. METHODS: Twenty seven rats were separated into 4 equal groups: Control, Cp (cisplatin, 6 mg/kg, intrapertoneally (ip)), F1 (cisplatin + 1 µg/day follistatin ip for 4 consecutive days) and F4 (cisplatin + 4 µg/day follistatin ip single dose) groups. Renal health was monitored by blood urea nitrogen, serum creatinine and histological analysis. Apoptosis, inflammation and oxidative stress was investigated in kidney tissue. Activin A levels in serum and kidney were evaluated as well. RESULTS: Follistatin administration showed a considerable nephroprotective effect against cisplatin-induced nephrotoxicity by preventing renal functional and structural abnormalities, apoptosis and inflammation. The activin A levels in both serum and kidney were also suppressed by follistatin administration. CONCLUSION: Exogenous follistatin ameliorates acute kidney injury, by blocking activin A. The renoprotective effect of follistatin against cisplatin-induced nephrotoxicity appears to be associated with its anti-inflammatory, antiapoptotic and direct nephroprotective actions (Tab. 1, Fig. 7, Ref. 23).

The caveolin-1 regulated protein follistatin protects against diabetic kidney disease.

Glomerular matrix protein accumulation, mediated largely by mesangial cells, is central to the pathogenesis of diabetic kidney disease. Our previous studies showed that the membrane microdomains caveolae and their marker protein caveolin-1 regulate matrix protein synthesis in mesangial cells in response to diabetogenic stimuli, and that caveolin-1 knockout mice are protected against diabetic kidney disease. In a screen to identify the molecular mechanism underlying this protection, we also established that secreted antifibrotic glycoprotein follistatin is significantly upregulated by caveolin-1 deletion. Follistatin potently neutralizes activins, members of the transforming growth factor-ß superfamily. A role for activins in diabetic kidney disease has not yet been established. Therefore, in vitro, we confirmed the regulation of follistatin by caveolin-1 in primary mesangial cells and showed that follistatin controls both basal and glucose-induced matrix production through activin inhibition. In vivo, we found activin A upregulation by immunohistochemistry in both mouse and human diabetic kidney disease. Importantly, administration of follistatin to type 1 diabetic Akita mice attenuated early diabetic kidney disease, characterized by albuminuria, hyperfiltration, basement membrane thickening, loss of endothelial glycocalyx and podocyte nephrin, and glomerular matrix accumulation. Thus, activin A is an important mediator of high glucose-induced profibrotic responses in mesangial cells, and follistatin may be a potential novel therapy for the prevention of diabetic kidney disease.

Follistatin-based ligand trap ACE-083 induces localized hypertrophy of skeletal muscle with functional improvement in models of neuromuscular disease.

Skeletal muscle is under inhibitory homeostatic regulation by multiple ligands of the transforming growth factor-ß (TGFß) superfamily. Follistatin is a secreted protein that promotes muscle growth and function by sequestering these ligands extracellularly. In the present study, we evaluated the potential of ACE-083 - a locally acting, follistatin-based fusion protein - as a novel therapeutic agent for focal or asymmetric myopathies. Characterization of ACE-083 in vitro revealed its high affinity for heparin and extracellular matrix while surface plasmon resonance and cell-based assays confirmed that ACE-083 binds and potently neutralizes myostatin, activin A, activin B and growth differentiation factor 11 (GDF11). Intramuscular administration of ACE-083 caused localized, dose-dependent hypertrophy of the injected muscle in wild-type mice and mouse models of Charcot-Marie-Tooth disease (CMT) and Duchenne muscular dystrophy, with no evidence of systemic muscle effects or endocrine perturbation. Importantly, ACE-083 also increased the force of isometric contraction in situ by the injected tibialis anterior muscle in wild-type mice and disease models and increased ankle dorsiflexion torque in CMT mice. Our results demonstrate the potential of ACE-083 as a therapeutic agent for patients with CMT, muscular dystrophy and other disorders with focal or asymmetric muscle atrophy or weakness.

Sarcopenia: Ammonia metabolism and hepatic encephalopathy.

Sarcopenia (loss of muscle mass and/or strength) frequently complicates liver cirrhosis and adversely affects the quality of life; cirrhosis related liver decompensation and significantly decreases wait-list and post-liver transplantation survival. The main therapeutic strategies to improve or reverse sarcopenia include dietary interventions (supplemental calorie and protein intake), increased physical activity (supervised resistance and endurance exercises), hormonal therapy (testosterone), and ammonia lowering agents (L-ornithine L-aspartate, branch chain amino acids) as well as mechanistic approaches that target underlying molecular and metabolic abnormalities. Besides other factors, hyperammonemia has recently gained attention and increase sarcopenia by various mechanisms including increased expression of myostatin, increased phosphorylation of eukaryotic initiation factor 2a, cataplerosis of α ketoglutarate, mitochondrial dysfunction, increased reactive oxygen species that decrease protein synthesis and increased autophagy-mediated proteolysis. Sarcopenia contributes to frailty and increases the risk of minimal and overt hepatic encephalopathy.

AAV-mediated follistatin gene therapy improves functional outcomes in the TIC-DUX4 mouse model of FSHD.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant or digenic disorder linked to derepression of the toxic DUX4 gene in muscle. There is currently no pharmacological treatment. The emergence of DUX4 enabled development of cell and animal models that could be used for basic and translational research. Since DUX4 is toxic, animal model development has been challenging, but progress has been made, revealing that tight regulation of DUX4 expression is critical for creating viable animals that develop myopathy. Here, we report such a model - the tamoxifen-inducible FSHD mouse model called TIC-DUX4. Uninduced animals are viable, born in Mendelian ratios, and overtly indistinguishable from WT animals. Induced animals display significant DUX4-dependent myopathic phenotypes at the molecular, histological, and functional levels. To demonstrate the utility of TIC-DUX4 mice for therapeutic development, we tested a gene therapy approach aimed at improving muscle strength in DUX4-expressing muscles using adeno-associated virus serotype 1.Follistatin (AAV1.Follistatin), a natural myostatin antagonist. This strategy was not designed to modulate DUX4 but could offer a mechanism to improve muscle weakness caused by DUX4-induced damage. AAV1.Follistatin significantly increased TIC-DUX4 muscle mass and strength even in the presence of DUX4 expression, suggesting that myostatin inhibition may be a promising approach to treat FSHD-associated weakness. We conclude that TIC-DUX4 mice are a relevant model to study DUX4 toxicity and, importantly, are useful in therapeutic development studies for FSHD.

Myostatin and activin blockade by engineered follistatin results in hypertrophy and improves dystrophic pathology in mdx mouse more than myostatin blockade alone.

BACKGROUND: Myostatin antagonists are being developed as therapies for Duchenne muscular dystrophy due to their strong hypertrophic effects on skeletal muscle. Engineered follistatin has the potential to combine the hypertrophy of myostatin antagonism with the anti-inflammatory and anti-fibrotic effects of activin A antagonism. METHODS: Engineered follistatin was administered to C57BL/6 mice for 4 weeks, and muscle mass and myofiber size was measured. In the mdx model, engineered follistatin was dosed for 12 weeks in two studies comparing to an Fc fusion of the activin IIB receptor or an anti-myostatin antibody. Functional measurements of grip strength and tetanic force were combined with tissue analysis for markers of necrosis, inflammation, and fibrosis to evaluate improvement in dystrophic pathology. RESULTS: In wild-type and mdx mice, dose-dependent increases in muscle mass and quadriceps myofiber size were observed for engineered follistatin. In mdx, increases in grip strength and tetanic force were combined with improvements in muscle markers for necrosis, inflammation, and fibrosis. Improvements in dystrophic pathology were greater for engineered follistatin than the anti-myostatin antibody. CONCLUSIONS: Engineered follistatin generated hypertrophy and anti-fibrotic effects in the mdx model.

Inhibition of activin signaling in lung adenocarcinoma increases the therapeutic index of platinum chemotherapy.

Resistance to platinum chemotherapy is a long-standing problem in the management of lung adenocarcinoma. Using a whole-genome synthetic lethal RNA interference screen, we identified activin signaling as a critical mediator of innate platinum resistance. The transforming growth factor-ß (TGFß) superfamily ligands activin A and growth differentiation factor 11 (GDF11) mediated resistance via their cognate receptors through TGFß-activated kinase 1 (TAK1), rather than through the SMAD family of transcription factors. Inhibition of activin receptor signaling or blockade of activin A and GDF11 by the endogenous protein follistatin overcame this resistance. Consistent with the role of activin signaling in acute renal injury, both therapeutic interventions attenuated acute cisplatin-induced nephrotoxicity, its major dose-limiting side effect. This cancer-specific enhancement of platinum-induced cell death has the potential to dramatically improve the safety and efficacy of chemotherapy in lung cancer patients.

Use Of Ankle Immobilization In Evaluating Treatments To Promote Longitudinal Muscle Growth In Mice.

INTRODUCTION: Difficulty in modeling congenital contractures (deformities of muscle-tendon unit development that include shortened muscles and lengthened tendons) has limited research of new treatments. METHODS: Early immobilization of the ankle in prepuberal mice was used to produce deformities similar to congenital contractures. Stretch treatment, electrostimulation, and local intramuscular injection of a follistatin analog (FST-288) were assessed as therapeutic interventions for these deformities. RESULTS: Ankle immobilization at full plantarflexion and 90 ° created tendon lengthening and muscle shortening in the tibialis anterior and soleus. Stretch treatment produced minimal evidence for longitudinal muscle growth and electrostimulation provided no additional benefit. Stretch treatment with FST-288 produced greater longitudinal muscle growth and less tendon lengthening, constituting the best treatment response. DISCUSSION: Ankle immobilization recapitulates key morphologic features of congenital contracture, and these features can be mitigated by a combination of stretch and pharmacological approaches that may be useful in patients. Muscle Nerve 58: 718-725, 2018.

Protein Engineering on Human Recombinant Follistatin: Enhancing Pharmacokinetic Characteristics for Therapeutic Application.

Follistatin (FS) is an important regulatory protein, a natural antagonist for transforming growth factor-ß family members activin and myostatin. The diverse biologic roles of the activin and myostatin signaling pathways make FS a promising therapeutic target for treating human diseases exhibiting inflammation, fibrosis, and muscle disorders, such as Duchenne muscular dystrophy. However, rapid heparin-mediated hepatic clearance of FS limits its therapeutic potential. We targeted the heparin-binding loop of FS for site-directed mutagenesis to improve clearance parameters. By generating a series of FS variants with one, two, or three negative amino acid substitutions, we demonstrated a direct and proportional relationship between the degree of heparin-binding affinity in vitro and the exposure in vivo. The triple mutation K(76,81,82)E abolished heparin-binding affinity, resulting in ∼20-fold improved in vivo exposure. This triple mutant retains full functional activity and an antibody-like pharmacokinetic profile, and shows a superior developability profile in physical stability and cell productivity compared with FS variants, which substitute the entire heparin-binding loop with alternative sequences. Our surgical approach to mutagenesis should also reduce the immunogenicity risk. To further lower this risk, we introduced a novel glycosylation site into the heparin-binding loop. This hyperglycosylated variant showed a 10-fold improved exposure and decreased clearance in mice compared with an IgG1 Fc fusion protein containing the native FS sequence. Collectively, our data highlight the importance of improving pharmacokinetic properties by manipulating heparin-binding affinity and glycosylation content and provide a valuable guideline to design desirable therapeutic FS molecules.

Follistatin treatment suppresses SERCA1b levels independently of other players of calcium homeostasis in C2C12 myotubes.

Follistatin (FS) is a high affinity activin-binding protein, neutralizing the effects of the Transforming Growth Factor-beta (TGF-ß) superfamily members, as myostatin (MSTN). Since MSTN emerged as a negative regulator, FS has been considered as a stimulator of skeletal muscle growth and differentiation. Here, we studied the effect of FS administration on the Ca2+-homeostasis of differentiating C2C12 skeletal muscle cells. FS-treatment increased the fusion index, the size of terminally differentiated myotubes, and transiently elevated the expression of the calcium-dependent protein phosphatase, calcineurin, at the beginning of differentiation. Functional experiments did not detect any alterations in the Ca2+ transients following the stimulation by KCl or caffeine in myotubes. On the other hand, decreased Ca2+-uptake capability was determined by calculating the maximal pump rate (332 ± 17 vs. 279 ± 11 µM/s, in control and FS-treated myotubes, respectively; p < 0.05). In the same way, the expression and ATPase activity of the neonatal sarcoplasmic/endoplasmic reticulum Ca2+ATPase (SERCA1b) were decreased (0.59 ± 0.01 vs. 0.19 ± 0.01 mM ATP/min, in control and FS-treated myotubes, respectively; p < 0.05). However, the expression level of other proteins involved in Ca2+-homeostasis and differentiation (calsequestrin, STIM1, MyoD) were not affected. Our results suggest that the FS controlled myotube growth is paralleled with the tight regulation of cytosolic calcium concentration, and the decline of SERCA1b appears to be one of the key components in this process.

The emerging role of Toll-like receptor 4 in myocardial inflammation.

Toll-like receptors (TLRs) are a family of pattern recognition receptors involved in cardiovascular diseases. Notably, numerous studies have demonstrated that TLR4 activates the expression of several of pro-inflammatory cytokine genes that play pivotal roles in myocardial inflammation, particularly myocarditis, myocardial infarction, ischemia-reperfusion injury, and heart failure. In addition, TLR4 is an emerging target for anti-inflammatory therapies. Given the significance of TLR4, it would be useful to summarize the current literature on the molecular mechanisms and roles of TLR4 in myocardial inflammation. Thus, in this review, we first introduce the basic knowledge of the TLR4 gene and describe the activation and signaling pathways of TLR4 in myocardial inflammation. Moreover, we highlight the recent progress of research on the involvement of TLR4 in myocardial inflammation. The information reviewed here may be useful to further experimental research and to increase the potential of TLR4 as a therapeutic target.

The effect of the DcpS inhibitor D156844 on the protective action of follistatin in mice with spinal muscular atrophy.

Spinal muscular atrophy (SMA), a leading genetic cause of pediatric death in the world, is an early-onset disease affecting the motor neurons in the anterior horn of the spinal cord. This degeneration of motor neurons leads to loss of muscle function. At the molecular level, SMA results from the loss of or mutation in the survival motor neuron 1 (SMN1) gene. The number of copies of the nearly duplicated gene SMN2 modulates the disease severity in humans as well as in transgenic mouse models for SMA. Most preclinical therapeutic trials focus on identifying ways to increase SMN2 expression and to alter its splicing. Other therapeutic strategies have investigated compounds which protect affected motor neurons and their target muscles in an SMN-independent manner. In the present study, the effect of a combination regimen of the SMN2 inducer D156844 and the protectant follistatin on the disease progression and survival was measured in the SMNΔ7 SMA mouse model. The D156844/follistatin combination treatment improved the survival of, delayed the end stage of disease in and ameliorated the growth rate of SMNΔ7 SMA mice better than follistatin treatment alone. The D156844/follistatin combination treatment, however, did not provide additional benefit over D156844 alone with respect to survival and disease end stage even though it provided some additional therapeutic benefit over D156844 alone with respect to motor phenotype.

Pharmacological inhibition of myostatin/TGF-ß receptor/pSmad3 signaling rescues muscle regenerative responses in mouse model of type 1 diabetes.

AIM: To study the influence of acute experimental diabetes on the regenerative potential of muscle stem (satellite) cells in mice. METHODS: Male C57BL/6 young mice were injected with a single dose of streptozotocin (STZ, 180 mg/kg, ip) to induce diabetes. The diabetic mice were treated with insulin (0.75 U/kg, ip), follistatin (12 µg/kg, im) or Alk5 inhibitor (5 µmol/L per kg, sc) once a day. On the first day when high glucose levels were found, cardiotoxin (CTX) was focally injected into tibialis anterior and gastronemius muscles of the mice. The muscles were harvested 3 d and 5 d after CTX injection, and myofibers and satellite cells were isolated. Quantitative ex-vivo and in-vivo assays of myogenic potential were used to evaluate the muscle regenerative responses. RESULTS: The satellite cells from the diabetic mice 3 d after CTX injection fail to activate, and the repair of muscle deteriorates, resembling that observed in old control mice. Furthermore, the satellite cells have excessive levels of myostatin, TGF-ß receptor 1, pSmad3 and the cell cycle inhibitor p15, while the level of TGF-ß1 remain unchanged. Treatment of the diabetic mice with insulin rescued muscle regenerative responses, and restored the expression levels of myostatin, TGF-ß receptor 1, pSmad3, and p15 to those similar of healthy controls. Treatment of the diabetic mice with the myostatin antagonist follistatin, or with the Alk5 inhibitor of TGF-ß receptor 1 (which did not diminish the blood glucose levels) rescued muscle regenerative responses and attenuated the myostatin/TGFß receptor/pSmad3 signaling. CONCLUSION: The muscle regenerative responses are incapacitated and repair of the tissue fails within hours after the initiation of hyperglycemia in a mouse model of type 1 diabetes, but stem cell function is rescued by insulin, as well as follistatin or an Alk5 inhibitor that blocks TGF-ß receptor signaling.

The activins and their binding protein, follistatin-Diagnostic and therapeutic targets in inflammatory disease and fibrosis.

The activins, as members of the transforming growth factor-ß superfamily, are pleiotrophic regulators of cell development and function, including cells of the myeloid and lymphoid lineages. Clinical and animal studies have shown that activin levels increase in both acute and chronic inflammation, and are frequently indicators of disease severity. Moreover, inhibition of activin action can reduce inflammation, damage, fibrosis and morbidity/mortality in various disease models. Consequently, activin A and, more recently, activin B are emerging as important diagnostic tools and therapeutic targets in inflammatory and fibrotic diseases. Activin antagonists such as follistatin, an endogenous activin-binding protein, offer considerable promise as therapies in conditions as diverse as sepsis, liver fibrosis, acute lung injury, asthma, wound healing and ischaemia-reperfusion injury.

Transgenic inactivation of murine myostatin does not decrease the severity of disease in a model of Spinal Muscular Atrophy.

Spinal Muscular Atrophy (SMA) is a devastating neurodegenerative disease and is a leading genetic cause of infantile death. SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1). The presence of a nearly identical copy gene called SMN2 has led to the development of several strategies that are designed to elevate SMN levels, and it is clear that SMN2 is an important modifier gene. However, the possibility exists that SMN-independent strategies to lessen the severity of the SMA phenotype could provide insight into disease development as well as aid in the identification of potential therapeutic targets. Muscle enhancement has been considered an interesting target for a variety of neurodegenerative diseases, including SMA. Previously we have shown in SMA mice that delivery of recombinant follistatin resulted in an extension in survival and a general lessening of disease severity. Follistatin is known to functionally block myostatin (MSTN), a potent inhibitor of muscle development. However, follistatin is a multifaceted protein involved in a variety of cellular pathways. To determine whether MSTN inhibition was the primary pathway associated with the previously reported follistatin results, we generated an animal model of SMA in which Mstn was genetically inactivated. In this report we characterize the novel SMA/Mstn model and demonstrate that Mstn inactivation does not significantly enhance muscle development in neonatal animals, nor does it result in an amelioration of the SMA phenotype.