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1.
Trop Biomed ; 38(1): 187-204, 2021 Mar 01.
Article in English | MEDLINE | ID: mdl-33797545

ABSTRACT

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.


Subject(s)
Activins/antagonists & inhibitors , Malaria/drug therapy , Tumor Necrosis Factor-alpha/blood , Activins/immunology , Animals , Cytokines/blood , Interferon-gamma/blood , Interleukin-10/blood , Malaria/immunology , Male , Mice , Mice, Inbred ICR , Plasmodium berghei
2.
Int J Mol Sci ; 22(6)2021 Mar 11.
Article in English | MEDLINE | ID: mdl-33799579

ABSTRACT

Diabetic kidney disease (DKD) is the leading cause of kidney failure. RhoA/Rho-associated protein kinase (ROCK) signaling is a recognized mediator of its pathogenesis, largely through mediating the profibrotic response. While RhoA activation is not feasible due to the central role it plays in normal physiology, ROCK inhibition has been found to be effective in attenuating DKD in preclinical models. However, this has not been evaluated in clinical studies as of yet. Alternate means of inhibiting RhoA/ROCK signaling involve the identification of disease-specific activators. This report presents evidence showing the activation of RhoA/ROCK signaling both in vitro in glomerular mesangial cells and in vivo in diabetic kidneys by two recently described novel pathogenic mediators of fibrosis in DKD, activins and cell-surface GRP78. Neither are present in normal kidneys. Activin inhibition with follistatin and neutralization of cell-surface GRP78 using a specific antibody blocked RhoA activation in mesangial cells and in diabetic kidneys. These data identify two novel RhoA/ROCK activators in diabetic kidneys that can be evaluated for their efficacy in inhibiting the progression of DKD.


Subject(s)
Activins/genetics , Diabetes Mellitus, Experimental/genetics , Heat-Shock Proteins/genetics , Mesangial Cells/metabolism , rhoA GTP-Binding Protein/genetics , Activins/antagonists & inhibitors , Activins/metabolism , Animals , Antibodies, Neutralizing/pharmacology , Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/metabolism , Diabetic Nephropathies/drug therapy , Diabetic Nephropathies/genetics , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/pathology , Disease Models, Animal , Endoplasmic Reticulum Chaperone BiP , Follistatin/pharmacology , Gene Expression Regulation , Heat-Shock Proteins/antagonists & inhibitors , Heat-Shock Proteins/metabolism , Humans , Male , Mesangial Cells/drug effects , Mesangial Cells/pathology , Mice , Mice, Inbred C57BL , Nephrectomy/methods , Primary Cell Culture , Signal Transduction , Streptozocin/administration & dosage , rho-Associated Kinases/genetics , rho-Associated Kinases/metabolism , rhoA GTP-Binding Protein/metabolism
3.
Methods Mol Biol ; 2273: 151-158, 2021.
Article in English | MEDLINE | ID: mdl-33604851

ABSTRACT

The first differentiation event in mammalian embryos is the formation of the trophectoderm, which is the progenitor of the outer epithelial component of the placenta and supports the fetus during intrauterine life. Our understanding of these events is limited, particularly in human, because of ethical and legal restrictions and availability of adequate in vitro models would be very advantageous. Here we describe a method that converts human fibroblasts into trophoblast-like cells, combining the use of 5-azacytidine-CR (5-aza-CR) to erase the original cell phenotype and a cocktail containing bone morphogenetic protein 4 (BMP4) with inhibitors of the Activin/Nodal/ERK signaling pathways, to drive erased fibroblasts into the trophoblastic differentiation. This innovative method uses very easily accessible cells to derive trophoblast-like cells and it can be useful to study embryo implantation disorders related to aging.


Subject(s)
Cell Culture Techniques/methods , Fibroblasts/cytology , Trophoblasts/cytology , Activins/antagonists & inhibitors , Animals , Azacitidine/pharmacology , Bone Morphogenetic Protein 4/metabolism , Bone Morphogenetic Protein 4/pharmacology , Cell Differentiation/drug effects , Cell Differentiation/genetics , Embryo Implantation , Embryo, Mammalian/metabolism , Embryonic Stem Cells/cytology , Female , Fibroblasts/drug effects , Fibroblasts/physiology , Humans , MAP Kinase Signaling System/drug effects , Mice , Nodal Protein/antagonists & inhibitors , Placenta/cytology , Pregnancy , Signal Transduction , Skin/cytology , Skin/growth & development
4.
Int J Biochem Cell Biol ; 131: 105902, 2021 02.
Article in English | MEDLINE | ID: mdl-33309621

ABSTRACT

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.


Subject(s)
Activins/pharmacology , Dual Oxidases/genetics , Myocytes, Cardiac/drug effects , Pyroptosis/drug effects , Reactive Oxygen Species/metabolism , Acetylcysteine/pharmacology , Activins/antagonists & inhibitors , Caspase 1/genetics , Caspase 1/metabolism , Coenzyme A Ligases/genetics , Coenzyme A Ligases/metabolism , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type III/genetics , Collagen Type III/metabolism , Dual Oxidases/antagonists & inhibitors , Dual Oxidases/metabolism , Free Radical Scavengers/pharmacology , Gene Expression Regulation , Humans , Interleukin-18/genetics , Interleukin-18/metabolism , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Oxidative Stress/drug effects , Primary Cell Culture , Pyroptosis/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Reactive Oxygen Species/agonists , Reactive Oxygen Species/antagonists & inhibitors , Signal Transduction , Smad2 Protein/genetics , Smad2 Protein/metabolism , Smad3 Protein/genetics , Smad3 Protein/metabolism
5.
J Clin Pharmacol ; 60(11): 1424-1431, 2020 11.
Article in English | MEDLINE | ID: mdl-32557665

ABSTRACT

We describe outcomes from the first-in-human study of garetosmab (a fully human monoclonal antibody that inhibits activin A) under development for the treatment of fibrodysplasia ossificans progressiva (FOP). In a double-blind, placebo-controlled phase 1 study, 40 healthy women of nonchildbearing potential were randomized to receive a single dose of intravenous garetosmab 0.3, 1, 3, or 10 mg/kg; subcutaneous garetosmab 300 mg; or placebo. Serum concentrations of functional garetosmab (with ≥1 arm free to bind to target), total activin A, and antidrug antibodies were measured predose and up to 113 days post-first dose. Garetosmab demonstrated an acceptable safety profile with no dose-limiting toxicities. Garetosmab displayed nonlinear pharmacokinetics with target-mediated elimination. With increasing doses of intravenous garetosmab, mean peak concentration increased in a dose-proportional manner; mean steady-state estimates ranged from 41.4 to 47.8 mL/kg. A greater than dose-proportional increase in mean area under the concentration-time curve from time zero extrapolated to infinity (range, 72.2-7520 mg*day/L) was observed, consistent with decreasing mean clearance (range, 4.35-1.34 mL/day/kg). Following administration of intravenous garetosmab, mean concentrations of total activin A increased in a dose-dependent manner. At 10 mg/kg, total activin A levels reached a state of little or no change between weeks 4 and 12, suggesting saturation of the target-mediated pathway. No safety signals were seen in this study to preclude investigation in patients. Following intravenous administration, garetosmab concentrations decreased quickly, then decreased over time (reflecting linear elimination), and finally decreased in a nonlinear phase, reflecting target-mediated elimination. Results here support further investigation. Garetosmab 10 mg/kg every 4 weeks intravenously is being evaluated in patients with FOP (NCT03188666).


Subject(s)
Activins/antagonists & inhibitors , Immunologic Factors/adverse effects , Immunologic Factors/pharmacokinetics , Activins/blood , Administration, Intravenous , Antibodies, Neutralizing , Area Under Curve , Double-Blind Method , Female , Healthy Volunteers , Humans , Immunologic Factors/administration & dosage , Immunologic Factors/blood , Injections, Subcutaneous
6.
Cancer Res ; 80(16): 3359-3371, 2020 08 15.
Article in English | MEDLINE | ID: mdl-32554750

ABSTRACT

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.


Subject(s)
Activin Receptors, Type I/metabolism , Activins/biosynthesis , Carcinoma, Pancreatic Ductal/etiology , Cellular Senescence/physiology , Pancreatic Neoplasms/etiology , Precancerous Conditions/etiology , Activin Receptors, Type I/genetics , Activin Receptors, Type II/metabolism , Activins/antagonists & inhibitors , Animals , Carcinoma, Pancreatic Ductal/metabolism , Disease Progression , Genes, ras , Humans , Mice , Pancreatic Neoplasms/metabolism , Phosphorylation , Precancerous Conditions/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , Transcriptional Activation
7.
Clin Transl Gastroenterol ; 11(5): e00152, 2020 05.
Article in English | MEDLINE | ID: mdl-32358238

ABSTRACT

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.


Subject(s)
Activins/metabolism , Anti-Inflammatory Agents/pharmacology , Pancreas/pathology , Pancreatitis/diagnosis , Severity of Illness Index , Activins/antagonists & inhibitors , Activins/blood , Activins/immunology , Animals , Anti-Inflammatory Agents/therapeutic use , Biomarkers/blood , Biomarkers/metabolism , Body Mass Index , Cell Line , Ceruletide/administration & dosage , Ceruletide/toxicity , Cohort Studies , Disease Models, Animal , Drug Evaluation, Preclinical , Female , Humans , Macrophage Activation/immunology , Macrophages , Mice , Pancreas/drug effects , Pancreas/immunology , Pancreatitis/blood , Pancreatitis/drug therapy , Pancreatitis/immunology , Patient Admission , Predictive Value of Tests
8.
Int J Oncol ; 57(1): 364-376, 2020 07.
Article in English | MEDLINE | ID: mdl-32377747

ABSTRACT

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.


Subject(s)
Activins/metabolism , Mouth Neoplasms/pathology , Neovascularization, Pathologic/pathology , Squamous Cell Carcinoma of Head and Neck/pathology , Vascular Endothelial Growth Factor A/metabolism , Activins/analysis , Activins/antagonists & inhibitors , Activins/genetics , Adult , Aged , Aged, 80 and over , Autocrine Communication/drug effects , Autocrine Communication/genetics , Cell Movement , Cell Proliferation , Female , Follistatin/pharmacology , Follistatin/therapeutic use , Gene Knockdown Techniques , Human Umbilical Vein Endothelial Cells , Humans , Male , Middle Aged , Mouth Mucosa/pathology , Mouth Neoplasms/blood supply , Mouth Neoplasms/drug therapy , Mouth Neoplasms/mortality , Paracrine Communication/drug effects , Paracrine Communication/genetics , Phosphorylation/drug effects , Phosphorylation/genetics , Prognosis , Protein Isoforms/metabolism , Smad2 Protein/metabolism , Smad3 Protein/metabolism , Squamous Cell Carcinoma of Head and Neck/blood supply , Squamous Cell Carcinoma of Head and Neck/drug therapy , Squamous Cell Carcinoma of Head and Neck/mortality
9.
Bioorg Med Chem Lett ; 30(3): 126892, 2020 02 01.
Article in English | MEDLINE | ID: mdl-31874826

ABSTRACT

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.


Subject(s)
Follistatin/chemistry , Myostatin/antagonists & inhibitors , Peptides/chemistry , Activins/antagonists & inhibitors , Activins/metabolism , Amino Acid Sequence , Animals , Binding Sites , Male , Mice , Mice, Inbred C57BL , Muscle, Skeletal/drug effects , Muscle, Skeletal/growth & development , Myostatin/metabolism , Peptides/metabolism , Peptides/pharmacology , Protein Binding , Structure-Activity Relationship , Transforming Growth Factor beta/antagonists & inhibitors , Transforming Growth Factor beta/metabolism
10.
Sci Signal ; 12(596)2019 08 27.
Article in English | MEDLINE | ID: mdl-31455725

ABSTRACT

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.


Subject(s)
Activins/antagonists & inhibitors , Follistatin/pharmacology , Neoplasm Proteins/antagonists & inhibitors , Neoplasms, Experimental/drug therapy , Signal Transduction/drug effects , Activins/metabolism , Animals , Killer Cells, Natural , Mice , Mice, Knockout , Neoplasm Proteins/metabolism , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology
11.
J Cachexia Sarcopenia Muscle ; 10(3): 662-686, 2019 06.
Article in English | MEDLINE | ID: mdl-30916493

ABSTRACT

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.


Subject(s)
Activins/antagonists & inhibitors , Aging/pathology , Muscle, Skeletal/pathology , Signal Transduction/drug effects , Wasting Syndrome/prevention & control , Activin Receptors, Type II/administration & dosage , Activin Receptors, Type II/genetics , Activins/metabolism , Animals , DNA-Binding Proteins/genetics , Disease Models, Animal , Endonucleases/genetics , Female , Humans , Injections, Intraperitoneal , Male , Mice , Mice, Transgenic , Muscle, Skeletal/drug effects , Myostatin/metabolism , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/genetics , Severity of Illness Index , Wasting Syndrome/diagnosis , Wasting Syndrome/genetics , Wasting Syndrome/pathology
12.
Skelet Muscle ; 8(1): 34, 2018 10 27.
Article in English | MEDLINE | ID: mdl-30368252

ABSTRACT

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.


Subject(s)
Activins/antagonists & inhibitors , Follistatin/therapeutic use , Muscular Dystrophies/drug therapy , Myostatin/antagonists & inhibitors , Animals , Follistatin/administration & dosage , Hand Strength , Male , Mice , Mice, Inbred C57BL , Mice, Inbred mdx , Muscle Contraction , Recombinant Proteins/administration & dosage , Recombinant Proteins/therapeutic use
13.
J Biol Chem ; 293(36): 14100-14111, 2018 09 07.
Article in English | MEDLINE | ID: mdl-30030373

ABSTRACT

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.


Subject(s)
Adipocytes/cytology , Cell Differentiation , Obesity , Proto-Oncogene Proteins/physiology , Signal Transduction , Activins/antagonists & inhibitors , Activins/metabolism , Adipose Tissue, White/growth & development , Animals , Mesenchymal Stem Cells/cytology , Mice , Smad2 Protein/antagonists & inhibitors
14.
Appl Immunohistochem Mol Morphol ; 26(2): 108-112, 2018 02.
Article in English | MEDLINE | ID: mdl-27389553

ABSTRACT

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.


Subject(s)
Breast Neoplasms/metabolism , Follistatin/metabolism , Mammary Glands, Human/physiology , Activins/antagonists & inhibitors , Breast Neoplasms/pathology , Carcinogenesis , Cell Cycle , Cell Proliferation , Disease Progression , Estrogen Receptor alpha/metabolism , Female , Follow-Up Studies , Humans , Immunohistochemistry , Menopause , Neoplasm Metastasis , Neoplasm Staging , Paraffin Embedding , Receptor, ErbB-2/metabolism
15.
Reproduction ; 154(3): 293-305, 2017 09.
Article in English | MEDLINE | ID: mdl-28667125

ABSTRACT

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.


Subject(s)
Apoptosis , Autoimmune Diseases/physiopathology , Disease Models, Animal , Follistatin/blood , Orchitis/physiopathology , Testis/metabolism , Up-Regulation , Activins/antagonists & inhibitors , Activins/metabolism , Animals , Autoimmune Diseases/immunology , Autoimmune Diseases/metabolism , Autoimmune Diseases/pathology , Biomarkers/blood , Biomarkers/metabolism , Blood-Testis Barrier/immunology , Blood-Testis Barrier/metabolism , Blood-Testis Barrier/pathology , Blood-Testis Barrier/physiopathology , Disease Progression , Fibrosis , Follistatin/administration & dosage , Follistatin/genetics , Follistatin/metabolism , Gene Expression Regulation , Gene Transfer Techniques , Inflammation Mediators/metabolism , Male , Mice, Inbred C57BL , Orchitis/immunology , Orchitis/metabolism , Orchitis/pathology , Recombinant Proteins/administration & dosage , Recombinant Proteins/blood , Recombinant Proteins/metabolism , Testis/immunology , Testis/pathology
16.
Proc Natl Acad Sci U S A ; 114(26): E5266-E5275, 2017 06 27.
Article in English | MEDLINE | ID: mdl-28607086

ABSTRACT

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.


Subject(s)
Dependovirus , Genetic Vectors , Muscle Proteins , Muscle, Skeletal/growth & development , Muscular Diseases , Signal Transduction , Transforming Growth Factor beta , Activins/antagonists & inhibitors , Activins/genetics , Activins/metabolism , Animals , Gene Targeting , Male , Mice , Muscle Proteins/antagonists & inhibitors , Muscle Proteins/genetics , Muscle Proteins/metabolism , Muscle, Skeletal/pathology , Muscular Diseases/genetics , Muscular Diseases/metabolism , Muscular Diseases/pathology , Organ Size/genetics , Smad Proteins/genetics , Smad Proteins/metabolism , Transforming Growth Factor beta/antagonists & inhibitors , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism
17.
Sci Rep ; 7(1): 2986, 2017 06 07.
Article in English | MEDLINE | ID: mdl-28592814

ABSTRACT

Maintenance of the adipose tissue requires a proper balance between self-renewal and differentiation of adipose progenitors (AP). Any deregulation leads either to fat overexpansion and obesity or fat loss and consequent lipodystrophies. Depending on the fat pad location, APs and adipocytes are heterogeneous. However, information on the pharmacological sensitivity of distinct APs to drugs known to alter the function of adipose tissue, especially HIV protease inhibitors (PIs) is scant. Here we show that PIs decreased proliferation and clonal expansion of APs, modifying their self-renewal potential. Lopinavir was the most potent PI tested. Decrease in self-renewal was accompanied by a reduced expression of the immediate early response gene IER3, a gene associated with tissue expansion. It was more pronounced in chin-derived APs than in knee-derived APs. Furthermore, lopinavir lowered the activin A-induced ERK1/2 phosphorylation. Expressions of the transcription factor EGR1 and its targets, including INHBA were subsequently altered. Therefore, activin A secretion was reduced leading to a dramatic impairment of APs self-renewal sustained by the activin A autocrine loop. All together, these observations highlight the activin A autocrine loop as a crucial effector to maintain APs self-renewal. Targeting this pathway by HIV-PIs may participate in the induction of unwanted side effects.


Subject(s)
Activins/antagonists & inhibitors , Adipose Tissue/cytology , Cell Proliferation/drug effects , HIV Protease Inhibitors/adverse effects , Lopinavir/adverse effects , Stem Cells/physiology , Cells, Cultured , Gene Expression Regulation/drug effects , Humans , Protein Interaction Maps/drug effects , Stem Cells/drug effects
18.
Nat Commun ; 8: 15153, 2017 04 28.
Article in English | MEDLINE | ID: mdl-28452368

ABSTRACT

Growth and differentiation factor 8 (GDF8) is a TGF-ß superfamily member, and negative regulator of skeletal muscle mass. GDF8 inhibition results in prominent muscle growth in mice, but less impressive hypertrophy in primates, including man. Broad TGF-ß inhibition suggests another family member negatively regulates muscle mass, and its blockade enhances muscle growth seen with GDF8-specific inhibition. Here we show that activin A is the long-sought second negative muscle regulator. Activin A specific inhibition, on top of GDF8 inhibition, leads to pronounced muscle hypertrophy and force production in mice and monkeys. Inhibition of these two ligands mimics the hypertrophy seen with broad TGF-ß blockers, while avoiding the adverse effects due to inhibition of multiple family members. Altogether, we identify activin A as a second negative regulator of muscle mass, and suggest that inhibition of both ligands provides a preferred therapeutic approach, which maximizes the benefit:risk ratio for muscle diseases in man.


Subject(s)
Activins/metabolism , Hypertrophy/pathology , Muscle Hypotonia/pathology , Muscle, Skeletal/growth & development , Myostatin/metabolism , Activin Receptors, Type II/metabolism , Activins/antagonists & inhibitors , Animals , Antibodies, Monoclonal/pharmacology , Body Mass Index , Dexamethasone/pharmacology , Humans , Isometric Contraction/physiology , Macaca fascicularis , Male , Mice , Mice, Inbred C57BL , Mice, SCID , Muscle, Skeletal/physiology , Myostatin/antagonists & inhibitors , Rats
19.
Stem Cells Transl Med ; 6(2): 490-501, 2017 02.
Article in English | MEDLINE | ID: mdl-28191760

ABSTRACT

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.


Subject(s)
Cell Differentiation , Cell Lineage , Cellular Reprogramming Techniques , Cellular Reprogramming , Epithelial Cells/physiology , Induced Pluripotent Stem Cells/physiology , Retinal Pigment Epithelium/physiology , Activins/antagonists & inhibitors , Activins/metabolism , Bone Morphogenetic Proteins/antagonists & inhibitors , Bone Morphogenetic Proteins/metabolism , Cell Differentiation/drug effects , Cell Lineage/drug effects , Cells, Cultured , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Gene Expression Regulation, Developmental , Humans , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Phenotype , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/metabolism , Signal Transduction , Time Factors , Transcriptome
20.
Transpl Int ; 30(1): 96-107, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27732750

ABSTRACT

Activins are members of the transforming growth factor-beta (TGF-ß) superfamily of cytokines. They play critical roles in the onset of acute and chronic inflammatory responses. The aim of this study was to investigate how activin inhibition affects acute kidney injury and inflammation after transplantation. The study was carried out in kidney transplantation and renal ischemia-reperfusion models in the rat. Soluble activin type 2 receptor (sActRIIB-Fc) was used to inhibit activin signaling. Transplantation groups were as follows: (i) cyclosporine A (CsA) (ii) CsA + sActRIIB-Fc, (iii) CsA+ inactive protein control Fc-G1. IRI groups were as follows: (i) no treatment, (ii) sActRIIB-Fc. Serum activin B concentration was significantly elevated after transplantation and IRI, whereas activin A was produced locally in renal allografts. Activin inhibition efficiently limited neutrophil, macrophage, and dendritic cell infiltration to the allografts measured 72 h after transplantation. In addition, sActRIIB-Fc treatment modulated serum cytokine response after transplantation and reduced the early accumulation of fibroblasts in the graft interstitium. In conclusion activin inhibition reduces the innate immune response early after renal transplantation in the rat. It also limits the accumulation of fibroblasts in the graft suggesting that activins may be involved in the fibrogenic signaling already early after kidney transplantation.


Subject(s)
Activins/antagonists & inhibitors , Allografts/immunology , Immunity, Innate , Kidney Transplantation , Kidney/immunology , Activins/metabolism , Animals , Cytokines/metabolism , Enzyme-Linked Immunosorbent Assay , Fibroblasts/metabolism , Humans , Inflammation , Male , Pilot Projects , Rats , Rats, Wistar , Renal Insufficiency/surgery , Reperfusion Injury , Signal Transduction , Time Factors , Transforming Growth Factor beta/metabolism , Transplantation, Homologous
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