Inhibition of Activin A suppressed tumor necrosis factor-α secretion and improved histopathological conditions in malarial mice.

Malaria infection still remains as one of the most prominent parasitic diseases afflicting mankind in tropical and subtropical regions. The severity of malaria infection has often been associated to exuberant host immune inflammatory responses that could possibly lead to severe immunopathological conditions and subsequent death of host tissues. Activin A is a protein belonging to the transforming growth factor-beta (TGF-ß) family that regulates multiple physiological processes and pathological-associated diseases. The biological roles of activin A have been associated with manipulation of inflammation-related processes and modulation of host immune responses. This implies that activin A protein could play a role in malaria pathogenesis since malaria infection has been closely linked to severe immune responses leading to death, However, the actual in vivo role of activin A in malaria infection remains elusive. Hence, this study was undertaken to investigate the involvement of activin A in malaria infection as well as to assess the modulating effects of activin A on the cytokine releases (TNF-α, IFN-γ and IL-10) and histopathological changes in major affected organs (kidney, liver, lung, brain and spleen) in malarial mice infected with Plasmodium berghei ANKA. Our results showed that the concentrations of plasma activin A were significantly increased in malarial mice throughout the study periods. Also. the systemic activin A level was positively correlated with malaria parasitemia. This indicates that activin A could play a role in malaria pathogenesis and malaria parasitemia development. Plasma TNF-α, IFN-γ and IL-10 cytokine levels were significantly increased in malarial mice at day-5 post infection, suggesting that these cytokines attributed to severe malaria pathogenesis. Histopathological features such as sequestration of parasitized red blood cells (pRBCs) and hemozoin formation were amongst the most common pathological conditions observed in tissues of major affected organs (kidney, liver, lung, brain and spleen) in malarial mice. Neutralization of activin A production via recombinant mouse activin RIIA Fc chimera (rmActivin RIIA Fc chimera) had significantly reduced the parasitemia levels in malarial mice. The release of TNF-α cytokine was significantly reduced as well as the sequestration of parasitized pRBCs and hemozoin formation in major affected organs in malarial mice were also alleviated following inhibition of activin A production. Overall, this preliminary study suggests that activin A could play an immune modulation role in malaria pathogenesis through modulation of TNF-α release that benefits host from severe pathological destructions provoked by intensified inflammatory responses. Further studies are warranted to elucidate the precise mechanism of immune modulation mediated by activin A and its associated immune-modulation mediators in regulating the inflammatory responses elicited during the course of malaria infection.

Knockdown of dual oxidase 1 suppresses activin A-induced fibrosis in cardiomyocytes via the reactive oxygen species-dependent pyroptotic pathway.

Fibrotic diseases account for more than 8 million deaths worldwide annually. Reactive oxygen species (ROS) has been shown to activate pyroptosis and promote the production of interleukin (IL)-1ß and IL-18, leading to fibrosis development. However, the role of dual oxidase 1 (DUOX1)-induced ROS production and pyroptosis in cardiac fibrosis remains largely unknown. Activin A was used to induce ROS and pyroptosis in cardiomyocytes. ROS level, pyroptosis, and cytokine production were detected using Active Oxygen Detection Kit, flow cytometry, and enzyme-linked immunosorbent assay, respectively. Western blotting analysis was used to measure expression changes of proteins. DUOX1 was silenced or overexpressed to investigate its role in fibrosis. We found that activin A induced ROS production and pyroptosis in cardiomyocytes, which was blocked by the ROS scavenger, N-acetyl-L-cysteine (NAC). Knockdown of DUOX1 reversed activin A-induced ROS production, pyroptosis, cytokine release, and the upregulation of proinflammatory proteins. Overexpression of DUOX1 resulted in opposite effects of knockdown DUOX1. Administration of an ROS scavenger blocked the effect of DUOX1 overexpression. Supplementation of IL-1ß and IL-18 caused significant fibrosis in human cardiac fibroblasts (hCFs). The knockdown of DUOX1 protected cardiomyocytes against activin A-induced fibrosis via the inhibition of ROS, cytokine release, and pyroptosis.

Oncogene-Induced Senescence Limits the Progression of Pancreatic Neoplasia through Production of Activin A.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly and aggressive cancer. Understanding mechanisms that drive preneoplastic pancreatic lesions is necessary to improve early diagnostic and therapeutic strategies. Mutations and inactivation of activin-like kinase (ALK4) have been demonstrated to favor PDAC onset. Surprisingly, little is known regarding the ligands that drive ALK4 signaling in pancreatic cancer or how this signaling pathway limits the initiation of neoplastic lesions. In this study, data mining and histologic analyses performed on human and mouse tumor tissues revealed that activin A is the major ALK4 ligand that drives PDAC initiation. Activin A, which is absent in normal acinar cells, was strongly induced during acinar-to-ductal metaplasia (ADM), which was promoted by pancreatitis or the activation of KrasG12D in mice. Activin A expression during ADM was associated with the cellular senescence program that is induced in precursor lesions. Blocking activin A signaling through the use of a soluble form of activin receptor IIB (sActRIIB-Fc) and ALK4 knockout in mice expressing KrasG12D resulted in reduced senescence associated with decreased expression of p21, reduced phosphorylation of H2A histone family member X (H2AX), and increased proliferation. Thus, this study indicates that activin A acts as a protective senescence-associated secretory phenotype factor produced by Kras-induced senescent cells during ADM, which limits the expansion and proliferation of pancreatic neoplastic lesions. SIGNIFICANCE: This study identifies activin A to be a beneficial, senescence-secreted factor induced in pancreatic preneoplastic lesions, which limits their proliferation and ultimately slows progression into pancreatic cancers.

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.

Activin A Modulates Inflammation in Acute Pancreatitis and Strongly Predicts Severe Disease Independent of Body Mass Index.

INTRODUCTION: Acute pancreatitis (AP) is a healthcare challenge with considerable mortality. Treatment is limited to supportive care, highlighting the need to investigate disease drivers and prognostic markers. Activin A is an established mediator of inflammatory responses, and its serum levels correlate with AP severity. We hypothesized that activin A is independent of body mass index (BMI) and is a targetable promoter of the AP inflammatory response. METHODS: We assessed whether BMI and serum activin A levels are independent markers to determine disease severity in a cohort of patients with AP. To evaluate activin A inhibition as a therapeutic, we used a cerulein-induced murine model of AP and treated mice with activin A-specific neutralizing antibody or immunoglobulin G control, both before and during the development of AP. We measured the production and release of activin A by pancreas and macrophage cell lines and observed the activation of macrophages after activin A treatment. RESULTS: BMI and activin A independently predicted severe AP in patients. Inhibiting activin A in AP mice reduced disease severity and local immune cell infiltration. Inflammatory stimulation led to activin A production and release by pancreas cells but not by macrophages. Macrophages were activated by activin A, suggesting activin A might promote inflammation in the pancreas in response to injury. DISCUSSION: Activin A provides a promising therapeutic target to interrupt the cycle of inflammation and tissue damage in AP progression. Moreover, assessing activin A and BMI in patients on hospital admission could provide important predictive measures for screening patients likely to develop severe disease.

Discovery of a follistatin-derived myostatin inhibitory peptide.

Follistatin is well known as an inhibitor of transforming growth factor (TGF)-ß superfamily ligands including myostatin and activin A. Myostatin, a negative regulator of muscle growth, is a promising target with which to treat muscle atrophic diseases. Here, we focused on the N-terminal domain (ND) of follistatin (Fst) that interacts with the type I receptor binding site of myostatin. Through bioassay of synthetic ND-derived fragment peptides, we identified DF-3, a new myostatin inhibitory 14-mer peptide which effectively inhibits myostatin, but fails to inhibit activin A or TGF-ß1, in an in vitro luciferase reporter assay. Injected intramuscularly, DF-3 significantly increases skeletal muscle mass in mice and consequently, it can serve as a platform for development of muscle enhancement based on myostatin inhibition.

Therapeutic blockade of activin-A improves NK cell function and antitumor immunity.

Natural killer (NK) cells are innate lymphocytes that play a major role in immunosurveillance against tumor initiation and metastatic spread. The signals and checkpoints that regulate NK cell fitness and function in the tumor microenvironment are not well defined. Transforming growth factor-ß (TGF-ß) is a suppressor of NK cells that inhibits interleukin-15 (IL-15)-dependent signaling events and increases the abundance of receptors that promote tissue residency. Here, we showed that NK cells express the type I activin receptor ALK4, which, upon binding to its ligand activin-A, phosphorylated SMAD2/3 to suppress IL-15-mediated NK cell metabolism. Activin-A impaired human and mouse NK cell proliferation and reduced the production of granzyme B to impair tumor killing. Similar to TGF-ß, activin-A also induced SMAD2/3 phosphorylation and stimulated NK cells to increase their cell surface expression of several markers of ILC1 cells. Activin-A also induced these changes in TGF-ß receptor-deficient NK cells, suggesting that activin-A and TGF-ß stimulate independent pathways that drive SMAD2/3-mediated NK cell suppression. Last, inhibition of activin-A by follistatin substantially slowed orthotopic melanoma growth in mice. These data highlight the relevance of examining TGF-ß-independent SMAD2/3 signaling mechanisms as a therapeutic axis to relieve NK cell suppression and promote antitumor immunity.

Compression of morbidity in a progeroid mouse model through the attenuation of myostatin/activin signalling.

BACKGROUND: One of the principles underpinning our understanding of ageing is that DNA damage induces a stress response that shifts cellular resources from growth towards maintenance. A contrasting and seemingly irreconcilable view is that prompting growth of, for example, skeletal muscle confers systemic benefit. METHODS: To investigate the robustness of these axioms, we induced muscle growth in a murine progeroid model through the use of activin receptor IIB ligand trap that dampens myostatin/activin signalling. Progeric mice were then investigated for neurological and muscle function as well as cellular profiling of the muscle, kidney, liver, and bone. RESULTS: We show that muscle of Ercc1Δ/- progeroid mice undergoes severe wasting (decreases in hind limb muscle mass of 40-60% compared with normal mass), which is largely protected by attenuating myostatin/activin signalling using soluble activin receptor type IIB (sActRIIB) (increase of 30-62% compared with untreated progeric). sActRIIB-treated progeroid mice maintained muscle activity (distance travel per hour: 5.6 m in untreated mice vs. 13.7 m in treated) and increased specific force (19.3 mN/mg in untreated vs. 24.0 mN/mg in treated). sActRIIb treatment of progeroid mice also improved satellite cell function especially their ability to proliferate on their native substrate (2.5 cells per fibre in untreated progeroids vs. 5.4 in sActRIIB-treated progeroids after 72 h in culture). Besides direct protective effects on muscle, we show systemic improvements to other organs including the structure and function of the kidneys; there was a major decrease in the protein content in urine (albumin/creatinine of 4.9 sActRIIB treated vs. 15.7 in untreated), which is likely to be a result in the normalization of podocyte foot processes, which constitute the filtration apparatus (glomerular basement membrane thickness reduced from 224 to 177 nm following sActRIIB treatment). Treatment of the progeric mice with the activin ligand trap protected against the development of liver abnormalities including polyploidy (18.3% untreated vs. 8.1% treated) and osteoporosis (trabecular bone volume; 0.30 mm3 in treated progeroid mice vs. 0.14 mm3 in untreated mice, cortical bone volume; 0.30 mm3 in treated progeroid mice vs. 0.22 mm3 in untreated mice). The onset of neurological abnormalities was delayed (by ~5 weeks) and their severity reduced, overall sustaining health without affecting lifespan. CONCLUSIONS: This study questions the notion that tissue growth and maintaining tissue function during ageing are incompatible mechanisms. It highlights the need for future investigations to assess the potential of therapies based on myostatin/activin blockade to compress morbidity and promote healthy ageing.

The regulatory protein SnoN antagonizes activin/Smad2 protein signaling and thereby promotes adipocyte differentiation and obesity in mice.

Ski-related oncogene SnoN (SnoN or SKIL) regulates multiple signaling pathways in a tissue- and developmental stage-dependent manner and has broad functions in embryonic angiogenesis, mammary gland alveologenesis, cancer, and aging. Here, we report that SnoN also plays a critical role in white adipose tissue (WAT) development by regulating mesenchymal stem cell (MSC) self-renewal and differentiation. We found that SnoN promotes MSC differentiation in the adipocyte lineage by antagonizing activin A/Smad2, but not TGFß/Smad3 signaling. Mice lacking SnoN or expressing a mutant SnoN defective in binding to the Smads were protected from high-fat diet-induced obesity and insulin resistance, and MSCs lacking a functional SnoN exhibited defective differentiation. We further demonstrated that activin, via Smad2, appears to be the major regulator of WAT development in vivo We also noted that activin A is abundantly expressed in WAT and adipocytes through an autocrine mechanism and promotes MSC self-renewal and inhibits adipogenic differentiation by inducing expression of the gene encoding the homeobox transcription factor Nanog. Of note, SnoN repressed activin/Smad2 signaling and activin A expression, enabling expression of adipocyte-specific transcription factors and promoting adipogenic differentiation. In conclusion, our study has revealed that SnoN plays an important in vivo role in adipocyte differentiation and WAT development in vivo by decreasing activity in the activin/Smad2 signaling pathway.

Follistatin Expression in Human Invasive Breast Tumors: Pathologic and Clinical Associations.

Follistatin is a potent native activin antagonist that is expressed in the normal mammary gland and in different breast proliferative diseases. Despite experimental evidence that follistatin can modulate the breast cancer cell cycle, the clinical significance of follistatin expression in these tumors is unknown. The aim of this study was to correlate the intensity of follistatin expression in invasive breast cancer with some of its clinical and pathologic features, such as the disease stage and the hormonal receptor status. Paraffin blocks of tumor samples that had been fixed in buffered formalin were obtained from 154 women subjected to surgery for breast cancer between 2008 and 2012. Sections from all paraffin blocks were cut and processed together by immunohistochemistry using a commercial monoclonal antibody to human follistatin. The intensity of follistatin staining was unrelated to the menopausal status, the disease stage, the grade, progesterone receptor expression, and local or systemic recurrence. However, follistatin immunoreactivity was significantly stronger in estrogen receptor (ER)-negative tumors than in ER-positive tumors. These findings suggest that follistatin expression in invasive breast cancer is unrelated to the disease severity and the risk of recurrence, but is more intense in ER-negative tumors.

Induction of experimental autoimmune orchitis in mice: responses to elevated circulating levels of the activin-binding protein, follistatin.

Experimental autoimmune orchitis (EAO) is a rodent model of chronic testicular inflammation that mimics the pathology observed in some types of human infertility. In a previous study, testicular expression of the inflammatory/immunoregulatory cytokine, activin A, was elevated in adult mice during the onset of EAO, indicating a potential role in the regulation of the disease. Consequently, we examined the development of EAO in mice with elevated levels of follistatin, an endogenous activin antagonist, as a potential therapeutic approach to testicular inflammation. Prior to EAO induction, mice received a single intramuscular injection of a non-replicative recombinant adeno-associated viral vector carrying a gene cassette of the circulating form of follistatin, FST315 (FST group). Serum follistatin levels were increased 5-fold in the FST group compared with the control empty vector (EV) group at 30 and 50 days of EAO, but intra-testicular levels of follistatin or activin A were not significantly altered. Induction of EAO was reduced, but not prevented, with mild-to-severe damage in 75% of the EV group and 40% of the FST group, at 50 days following immunisation with testicular homogenate. However, the EAO damage score (based on disruption of the blood-testis barrier, apoptosis, testicular damage and fibrosis) and extent of intratesticular inflammation (expression of inflammatory mediators) were directly proportional to the levels of activin A measured in the testis at 50 days. These data implicate activin A in the progression of EAO, thereby providing a potential therapeutic target; however, elevating circulating follistatin levels were not sufficient to prevent EAO development.

Specific targeting of TGF-ß family ligands demonstrates distinct roles in the regulation of muscle mass in health and disease.

The transforming growth factor-ß (TGF-ß) network of ligands and intracellular signaling proteins is a subject of intense interest within the field of skeletal muscle biology. To define the relative contribution of endogenous TGF-ß proteins to the negative regulation of muscle mass via their activation of the Smad2/3 signaling axis, we used local injection of adeno-associated viral vectors (AAVs) encoding ligand-specific antagonists into the tibialis anterior (TA) muscles of C57BL/6 mice. Eight weeks after AAV injection, inhibition of activin A and activin B signaling produced moderate (∼20%), but significant, increases in TA mass, indicating that endogenous activins repress muscle growth. Inhibiting myostatin induced a more profound increase in muscle mass (∼45%), demonstrating a more prominent role for this ligand as a negative regulator of adult muscle mass. Remarkably, codelivery of activin and myostatin inhibitors induced a synergistic response, resulting in muscle mass increasing by as much as 150%. Transcription and protein analysis indicated that this substantial hypertrophy was associated with both the complete inhibition of the Smad2/3 pathway and activation of the parallel bone morphogenetic protein (BMP)/Smad1/5 axis (recently identified as a positive regulator of muscle mass). Analyses indicated that hypertrophy was primarily driven by an increase in protein synthesis, but a reduction in ubiquitin-dependent protein degradation pathways was also observed. In models of muscular dystrophy and cancer cachexia, combined inhibition of activins and myostatin increased mass or prevented muscle wasting, respectively, highlighting the potential therapeutic advantages of specifically targeting multiple Smad2/3-activating ligands in skeletal muscle.

Directing Differentiation of Pluripotent Stem Cells Toward Retinal Pigment Epithelium Lineage.

Development of efficient and reproducible conditions for directed differentiation of pluripotent stem cells into specific cell types is important not only to understand early human development but also to enable more practical applications, such as in vitro disease modeling, drug discovery, and cell therapies. The differentiation of stem cells to retinal pigment epithelium (RPE) in particular holds promise as a source of cells for therapeutic replacement in age-related macular degeneration. Here we show development of an efficient method for deriving homogeneous RPE populations in a period of 45 days using an adherent, monolayer system and defined xeno-free media and matrices. The method utilizes sequential inhibition and activation of the Activin and bone morphogenetic protein signaling pathways and can be applied to both human embryonic stem cells and induced pluripotent stem cells as the starting population. In addition, we use whole genome transcript analysis to characterize cells at different stages of differentiation that provides further understanding of the developmental dynamics and fate specification of RPE. We show that with the described method, RPE develop through stages consistent with their formation during embryonic development. This characterization- together with the absence of steps involving embryoid bodies, three-dimensional culture, or manual dissections, which are common features of other protocols-makes this process very attractive for use in research as well as for clinical applications. Stem Cells Translational Medicine 2017;6:490-501.

New expectations in the treatment of anemia in chronic kidney disease. / Nuevas expectativas en el tratamiento de la anemia en la enfermedad renal crónica.

The new drugs developed for the treatment of anemia in chronic kidney disease patients, together with their mechanisms of action are reviewed. At present, many of them are already in advanced stages of clinical trials and is expected to be incorporated into the therapeutic arsenal in the coming years. The potential benefits and possible limitations are also described.

Transforming Growth Factor-ß Family Ligands Can Function as Antagonists by Competing for Type II Receptor Binding.

Transforming growth factor-ß (TGF-ß) family ligands are pleiotropic cytokines. Their physiological activities are not determined by a simple coupling of stimulus and response, but depend critically on context, i.e. the interplay of receptors, ligands, and regulators that form the TGF-ß signal transduction system of a cell or tissue. How these different components combine to regulate signaling activities remains poorly understood. Here, we describe a ligand-mediated mechanism of signaling regulation. Based on the observation that the type II TGF-ß family receptors ActRIIA, ActRIIB, and BMPRII interact with a large group of overlapping ligands at overlapping epitopes, we hypothesized high affinity ligands compete with low affinity ligands for receptor binding and signaling. We show activin A and other high affinity ligands directly inhibited signaling by the low affinity ligands BMP-2, BMP-7, and BMP-9. We demonstrate activin A functions as a competitive inhibitor that blocks the ligand binding epitope on type II receptors. We propose binding competition and signaling antagonism are integral functions of the TGF-ß signal transduction system. These functions could help explain how activin A modulates physiological signaling during extraordinary cellular responses, such as injury and wound healing, and how activin A could elicit disease phenotypes such as cancer-related muscle wasting and fibrosis.

Inhibition of Activin Signaling Slows Progression of Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD), characterized by the formation of numerous kidney cysts, is caused by PKD1 or PKD2 mutations and affects 0.1% of the population. Although recent clinical studies indicate that reduction of cAMP levels slows progression of PKD, this finding has not led to an established safe and effective therapy for patients, indicating the need to find new therapeutic targets. The role of TGF-ß in PKD is not clearly understood, but nuclear accumulation of phosphorylated SMAD2/3 in cyst-lining cells suggests the involvement of TGF-ß signaling in this disease. In this study, we ablated the TGF-ß type 1 receptor (also termed activin receptor-like kinase 5) in renal epithelial cells of PKD mice, which had little to no effect on the expression of SMAD2/3 target genes or the progression of PKD. Therefore, we investigated whether alternative TGF-ß superfamily ligands account for SMAD2/3 activation in cystic epithelial cells. Activins are members of the TGF-ß superfamily and drive SMAD2/3 phosphorylation via activin receptors, but activins have not been studied in the context of PKD. Mice with PKD had increased expression of activin ligands, even at early stages of disease. In addition, treatment with a soluble activin receptor IIB fusion (sActRIIB-Fc) protein, which acts as a soluble trap to sequester activin ligands, effectively inhibited cyst formation in three distinct mouse models of PKD. These data point to activin signaling as a key pathway in PKD and a promising target for therapy.

Experimental and investigational therapies for chemotherapy-induced anemia.

INTRODUCTION: In cancer patients, anemia is frequently observed, particularly as a consequence to chemotherapy (chemotherapy-induced anemia, CIA). CIA is treated with Red Blood Cell transfusions and erythropoiesis-stimulating agents (ESAs). However, the use of ESAs in anemic cancer patients is associated with reduced survival time and time to progression. Consequently, new therapeutic options are needed. AREAS COVERED: In this article, the authors discuss new erythroid-enhancing agents (EEAs) that act differently to erythropoietin. Specifically, the article summarizes the early clinical development of activin antagonists (Sotatercep [ACE-011] and ACE-536) and hepcidin antagonists [NOX-H94]). EXPERT OPINION: Both Activin RIIA trap agents and hepcidin inhibitors are promising new EEAs, but their safety profile, and their impact on treating CIA, needs to be carefully assessed in controlled clinical trials over longer periods of time. It is also important to carefully evaluate CIA patients to properly assess the physiopathological mechanisms responsible for the development of their anemic condition and provide patients with the most appropriate treatment plan.

Virtual High-Throughput Screening To Identify Novel Activin Antagonists.

Activin belongs to the TGFß superfamily, which is associated with several disease conditions, including cancer-related cachexia, preterm labor with delivery, and osteoporosis. Targeting activin and its related signaling pathways holds promise as a therapeutic approach to these diseases. A small-molecule ligand-binding groove was identified in the interface between the two activin ßA subunits and was used for a virtual high-throughput in silico screening of the ZINC database to identify hits. Thirty-nine compounds without significant toxicity were tested in two well-established activin assays: FSHß transcription and HepG2 cell apoptosis. This screening workflow resulted in two lead compounds: NUCC-474 and NUCC-555. These potential activin antagonists were then shown to inhibit activin A-mediated cell proliferation in ex vivo ovary cultures. In vivo testing showed that our most potent compound (NUCC-555) caused a dose-dependent decrease in FSH levels in ovariectomized mice. The Blitz competition binding assay confirmed target binding of NUCC-555 to the activin A:ActRII that disrupts the activin A:ActRII complex's binding with ALK4-ECD-Fc in a dose-dependent manner. The NUCC-555 also specifically binds to activin A compared with other TGFß superfamily member myostatin (GDF8). These data demonstrate a new in silico-based strategy for identifying small-molecule activin antagonists. Our approach is the first to identify a first-in-class small-molecule antagonist of activin binding to ALK4, which opens a completely new approach to inhibiting the activity of TGFß receptor superfamily members. in addition, the lead compound can serve as a starting point for lead optimization toward the goal of a compound that may be effective in activin-mediated diseases.

The activin A antagonist follistatin inhibits cystic fibrosis-like lung inflammation and pathology.

Cystic fibrosis (CF) is the most common life-limiting genetically acquired respiratory disorder. Patients with CF have thick mucus obstructing the airways leading to recurrent infections, bronchiectasis and neutrophilic airway inflammation culminating in deteriorating lung function. Current management targets airway infection and mucus clearance, but despite recent advances in care, life expectancy is still only 40 years. We investigated whether activin A is elevated in CF lung disease and whether inhibiting activin A with its natural antagonist follistatin retards lung disease progression. We measured serum activin A levels, lung function and nutritional status in CF patients. We studied the effect of activin A on CF lung pathogenesis by treating newborn CF transgenic mice (ß-ENaC) intranasally with the natural activin A antagonist follistatin. Activin A levels were elevated in the serum of adult CF patients, and correlated inversely with lung function and body mass index. Follistatin treatment of newborn ß-ENaC mice, noted for respiratory pathology mimicking human CF, decreased the airway activin A levels and key features of CF lung disease including mucus hypersecretion, airway neutrophilia and levels of mediators that regulate inflammation and chemotaxis. Follistatin treatment also increased body weight and survival of ß-ENaC mice, with no evidence of local or systemic toxicity. Our findings demonstrate that activin A levels are elevated in CF and provide proof-of-concept for the use of the activin A antagonist, follistatin, as a therapeutic in the long-term management of lung disease in CF patients.

Development of novel activin-targeted therapeutics.

Soluble activin type II receptors (ActRIIA/ActRIIB), via binding to diverse TGF-ß proteins, can increase muscle and bone mass, correct anemia or protect against diet-induced obesity. While exciting, these multiple actions of soluble ActRIIA/IIB limit their therapeutic potential and highlight the need for new reagents that target specific ActRIIA/IIB ligands. Here, we modified the activin A and activin B prodomains, regions required for mature growth factor synthesis, to generate specific activin antagonists. Initially, the prodomains were fused to the Fc region of mouse IgG2A antibody and, subsequently, "fastener" residues (Lys(45), Tyr(96), His(97), and Ala(98); activin A numbering) that confer latency to other TGF-ß proteins were incorporated. For the activin A prodomain, these modifications generated a reagent that potently (IC(50) 5 nmol/l) and specifically inhibited activin A signaling in vitro, and activin A-induced muscle wasting in vivo. Interestingly, the modified activin B prodomain inhibited both activin A and B signaling in vitro (IC(50) ~2 nmol/l) and in vivo, suggesting it could serve as a general activin antagonist. Importantly, unlike soluble ActRIIA/IIB, the modified prodomains did not inhibit myostatin or GDF-11 activity. To underscore the therapeutic utility of specifically antagonising activin signaling, we demonstrate that the modified activin prodomains promote significant increases in muscle mass.