Activin A as a potential biomarker for preserved ratio impaired spirometry (PRISm) and clinical outcomes in community-dwelling adults.

INTRODUCTION: This study endeavors to decipher the association between Activin A and PRISm, thereby addressing the potential of Activin A as a serum biomarker for early detection and long-term clinical outcome prediction of PRISm and subsequent all-cause mortality. METHODS: The study sample comprised middle-aged and older adults from the I-Lan Longitudinal Aging Study. Pulmonary function including forced vital capacity (FVC) and forced expiratory volume in one second (FEV1) were measured. Demographic data and laboratory data (including serum Activin A levels) were also collected. Multivariate logistic regression and Cox proportional hazards models were used to identify independent predictors of PRISm and all-cause mortality, respectively. RESULTS: Among 711 eligible participants, 34 % had PRISm. The risk of PRISm elevated with Activin A levels in group quartiles (adjusted odds ratio (aOR), Q2: 1.606 [95 % CI 0.972-2.652], p = 0.064, Q3: 2.666 [1.635-4.348], p < 0.001, Q4: 3.225 [1.965-5.293], p < 0.001). On the other hand, lower hemoglobin (aOR: 1.122, p = 0.041) and higher blood urea nitrogen (BUN) levels (aOR: 1.033, p = 0.048) were associated with increased risk of PRISm. In addition, the PRISm group had a higher all-cause mortality rate (non-PRISm 4.5% vs. PRISm 8.3 %, p = 0.038). Multivariate Cox models also identify a higher level of Activin A as a risk factor of all-cause mortality (aHR: 1.001 [1.000-1.003], p = 0.042). CONCLUSIONS: Higher Activin A quartiles were linked to increased risk of PRISm, along with lower hemoglobin and higher BUN levels. Additonally, elevated Activin A was a significant risk factor of all-cause mortality.

Activin Signaling Pathway Specialization During Embryonic and Skeletal Muscle Development in Rainbow Trout (Oncorhynchus mykiss).

Activin signaling is essential for proper embryonic, skeletal muscle, and reproductive development. Duplication of the pathway in teleost fish has enabled diversification of gene function across the pathway but how gene duplication influences the function of activin signaling in non-mammalian species is poorly understood. Full characterization of activin receptor signaling pathway expression was performed across embryonic development and during early skeletal muscle growth in rainbow trout (RBT, Oncorhynchus mykiss). Rainbow trout are a model salmonid species that have undergone two additional rounds of whole genome duplication. A small number of genes were expressed early in development and most genes increased expression throughout development. There was limited expression of activin Ab in RBT embryos despite these genes exhibiting significantly elevated expression in post-hatch skeletal muscle. CRISPR editing of the activin Aa1 ohnolog and subsequent production of meiotic gynogenetic offspring revealed that biallelic disruption of activin Aa1 did not result in developmental defects, as occurs with knockout of activin A in mammals. The majority of gynogenetic offspring exhibited homozygous activin Aa1 genotypes (wild type, in-frame, or frameshift) derived from the mosaic founder female. The research identifies mechanisms of specialization among the duplicated activin ohnologs across embryonic development and during periods of high muscle growth in larval and juvenile fish. The knowledge gained provides insights into potential viable gene-targeting approaches for engineering the activin receptor signaling pathway and establishes the feasibility of employing meiotic gynogenesis as a tool for producing homozygous F1 genome-edited fish for species with long-generation times, such as salmonids.

Pathological insights into activin A: Molecular underpinnings and therapeutic prospects in various diseases.

Activin A (Act A) is a member of the TGFß (transforming growth factor ß) superfamily. It communicates via the Suppressor of Mothers against Decapentaplegic Homolog (SMAD2/3) proteins which govern processes such as cell proliferation, wound healing, apoptosis, and metabolism. Act A produces its action by attaching to activin receptor type IIA (ActRIIA) or activin receptor type IIB (ActRIIB). Increasing circulating Act A increases ActRII signalling, which on phosphorylation initiates the ALK4 (activin receptor-like kinase 4) type 1 receptor which further turns on the SMAD pathway and hinders cell functioning. Once triggered, this route leads to gene transcription, differentiation, apoptosis, and extracellular matrix (ECM) formation. Act A also governs the immunological and inflammatory responses of the body, as well as cell death. Moreover, Act A levels have been observed to elevate in several disorders like renal fibrosis, CKD, asthma, NAFLD, cardiovascular diseases, cancer, inflammatory conditions etc. Here, we provide an update on the recent studies relevant to the role of Act A in the modulation of various pathological disorders, giving an overview of the biology of Act A and its signalling pathways, and discuss the possibility of incorporating activin-A targeting as a novel therapeutic approach for the control of various disorders. Pathways such as SMAD signaling, in which SMAD moves to the nucleus by making a complex and leads to tissue fibrosis in CKD, STAT3, which drives renal fibroblast activity and the production of ECM, Kidney injury molecule (KIM-1) in the synthesis, deposition of ECM proteins, SERCA2a (sarcoplasmic reticulum Ca2+ ATPase) in cardiac dysfunction, and NF-κB (Nuclear factor kappa-light-chain-enhancer of activated B cells) in inflammation are involved in Act A signaling, have also been discussed.

The comparative effect of teriparatide and denosumab on activins, follistatins and inhibins in women with postmenopausal osteoporosis.

The activins-follistatins-inhibins (AFI) hormonal system affects bone metabolism. Treatments that alter bone metabolism may also alter the AFI molecules. In this non-randomized, open-label, head-to-head comparative study, circulating levels of the AFI system were evaluated in postmenopausal women with osteoporosis treated for 12 months with either teriparatide (n = 23) or denosumab (n = 22). Τeriparatide treatment increased activin B (p = 0.01) and activin AB (p = 0.004) and the ratios activin A/follistatin (p = 0.006), activin B/follistatin (p = 0.007), activin AB/follistatin (p < 0.001) and activin AB/FSTL3 (p = 0.034). The significant p for trend in group*time interactions of activins B and AB and of the ratio activin AB/FSTL3 remained robust after adjustment for body mass index (BMI) and lumbar spine bone mineral density (LS BMD) but it was lost for activin B after adjustment for previous antiresorptive treatment. The effect of teriparatide on BMD was attenuated when it was adjusted for baseline activins levels or their 12-month changes. No changes were observed after denosumab treatment. In conclusion, activins B and AB, as well as the ratios of all activins to follistatin and of activin AB to FSTL3 increased with teriparatide treatment, possibly in a compensatory manner. Future studies are needed to study the potentially important role activins may play in bone biology and any associations with the effect of teriparatide on BMD.

Bone and the muscle, comprise two tissues that are considered to interact with each other, not only through mechanical but also through endocrine signals. Several components of the activins-follistatins-inhibins (AFI) hormonal system have been shown to be secreted by the muscle and affect the bone possibly contributing to this interplay. We have previously investigated levels of the AFI molecules in case­control studies and reported differences between osteoporotic versus osteopenic versus postmenopausal and premenopausal women with normal bone mineral density (BMD). In this 12-month, non-randomized, open-labeled, head-to-head comparative study, we prospectively compared the effect of antiosteoporotic agents with opposite effect on bone metabolism, i.e., teriparatide versus denosumab, on the circulating concentrations of all known molecules of the AFI system in postmenopausal women with osteoporosis. We observed increases of activins after teriparatide treatment, but no effect after denosumab treatment on any of the AFI molecules studied. Since activins are mainly acting in an autocrine way and since activin B and AB have not been extensively studied, further studies in the basic research, preclinical and clinical research fields are required to expand these observations and fully elucidate physiology and any therapeutic potential.

Contemporary Treatment of Pulmonary Arterial Hypertension: A US Perspective.

Pulmonary arterial hypertension (PAH) is a complex fatal condition which requires aggressive treatment with close monitoring. Significant progress has been made over the last three decades in the treatment of PAH but despite this progress, survival has remained unacceptably low. In the quest to improve survival, therapeutic interventions play a central role. In the last few years, there have been remarkable attempts to identify novel treatments. Finally, we have had a breakthrough with the discovery of the fourth treatment pathway in PAH. Activin signaling inhibition distinguishes itself as a potential antiproliferative intervention as opposed to the traditional therapies which mediate their effect primarily by vasodilatation. With this novel treatment pathway, we stand at an important milestone with an exciting future ahead and the natural question of when to utilize Activin signaling inhibitor (ASI) for the treatment of PAH. In this state-of-the-art review, we focus on the placement of this novel agent in the PAH treatment paradigm based on the available evidence, with special focus on the US patient population. This review also provides an expert opinion of the current treatment algorithm on important subgroups of patients with comorbidities from the US perspective.

Activin A Is a Master Regulator of Phenotypic Switch in Adipose Stromal Cells Initiated by Activated Immune Cell-Secreted Interleukin-1ß.

Prolonged tissue ischemia and inflammation lead to organ deterioration and are often accompanied by microvasculature rarefaction, fibrosis, and elevated systemic Activin A (ActA), the level of which frequently correlates with disease severity. Mesenchymal stromal cells are prevalent in the perivascular niche and are likely involved in tissue homeostasis and pathology. This study investigated the effects of inflammatory cells on modulation of phenotype of adipose mesenchymal stromal cells (ASC) and the role of ActA in this process. Peripheral blood mononuclear cells were activated with lipopolysaccharide (activated peripheral blood mononuclear cells [aPBMC]) and presented to ASC. Expression of smooth muscle/myofibroblast markers, ActA, transforming growth factors beta 1-3 (TGFß1-3), and connective tissue growth factor (CTGF) was assessed in ASC. Silencing approaches were used to dissect the signaling cascade of aPBMC-induced acquisition of myofibroblast phenotype by ASC. ASC cocultured with aPBMC or exposed to the secretome of aPBMC upregulated smooth muscle cell markers alpha smooth muscle actin (αSMA), SM22α, and Calponin I; increased contractility; and initiated expression of ActA. Interleukin (IL)-1ß was sufficient to replicate this response, whereas blocking IL-1ß eliminated aPBMC effects. ASC-derived ActA stimulated CTGF and αSMA expression in ASC; the latter independent of CTGF. Induction of αSMA in ASC by IL-1ß or ActA-enriched media relied on extracellular enzymatic activity. ActA upregulated mRNA levels of several extracellular matrix proteins in ASC, albeit to a lesser degree than TGFß1, and marginally increased cell contractility. In conclusion, the study suggests that aPBMC induce myofibroblast phenotype with weak fibrotic activity in perivascular progenitors, such as ASC, through the IL-1ß-ActA signaling axis, which also promotes CTGF secretion, and these effects require ActA extracellular enzymatic processing.

The placenta in fetal death: Molecular evidence of dysregulation of inflammatory, proliferative and fetal protective pathways.

BACKGROUND: It is estimated that over 2 million cases of fetal death occur worldwide every year, but, despite the high incidence, several basic and clinical characteristics of this disorder are still unclear. Placenta is suggested to play a central role in fetal death. Placenta produces hormones, cytokines and growth factors that modulate functions of the placental-maternal unit. Fetal death has been correlated with impaired secretion of some of these regulatory factors. OBJECTIVE(S): The aim of the present study was to evaluate, in placentas collected from fetal death, the gene expression of inflammatory, proliferative and protective factors. STUDY DESIGN: Cases of fetal death in singleton pregnancy were retrospectively selected, excluding pregnancies complicated by fetal anomalies, gestational diabetes, intrauterine growth restriction and moderate to severe maternal diseases. A group of placentas collected from healthy singleton term pregnancies were used as controls. Groups were compared regarding maternal and gestational age, fetal sex and birth weight. Placental mRNA expression of inflammatory (IL-6), proliferative (Activin A, TGF-ß1) and regulatory (VEGF, VEGFR2, ATP-binding cassette (ABC) transporters ABCB1 and ABCG2, sphingosine 1-phosphate (S1P) signaling pathway) markers was conducted using real-time PCR. Statistical analysis and graphical representation of the data were performed using the GraphPad Prism 5 software. For the statistical analysis, Student's t-test was used, and P values < 0.05 were considered significant. RESULTS: Placental mRNA expression of IL-6 and VEGFR2 resulted significantly higher in the fetal death group compared to controls (P<0.01), while activin A, ABCB1 and ABCG2 expression resulted significantly lower (P<0.01). A significant alteration in the S1P signaling pathway was found in the fetal death group, with an increased expression of the specific receptor isoforms sphingosine 1-phosphate receptor 1, 3 and 4 (S1P1, S1P3, S1P4) and of sphingosine kinase 2 (SK2), one of the enzyme isoforms responsible for S1P synthesis (P<0.01). CONCLUSION: (s): The present study confirmed a significantly increased expression of placental IL-6 and VEGFR2 mRNA, and for the first time showed an increased expression of S1P receptors and SK2 as well as a decreased expression of activin A and of selected ATP-binding cassette transporters, suggesting that multiple inflammatory and protective factors are deranged in placenta of fetal death.

A Lifelike guided journey through the pathophysiology of pulmonary hypertension-from measured metabolites to the mechanism of action of drugs.

Introduction: Pulmonary hypertension (PH) is a pathological condition that affects approximately 1% of the population. The prognosis for many patients is poor, even after treatment. Our knowledge about the pathophysiological mechanisms that cause or are involved in the progression of PH is incomplete. Additionally, the mechanism of action of many drugs used to treat pulmonary hypertension, including sotatercept, requires elucidation. Methods: Using our graph-powered knowledge mining software Lifelike in combination with a very small patient metabolite data set, we demonstrate how we derive detailed mechanistic hypotheses on the mechanisms of PH pathophysiology and clinical drugs. Results: In PH patients, the concentration of hypoxanthine, 12(S)-HETE, glutamic acid, and sphingosine 1 phosphate is significantly higher, while the concentration of L-arginine and L-histidine is lower than in healthy controls. Using the graph-based data analysis, gene ontology, and semantic association capabilities of Lifelike, led us to connect the differentially expressed metabolites with G-protein signaling and SRC. Then, we associated SRC with IL6 signaling. Subsequently, we found associations that connect SRC, and IL6 to activin and BMP signaling. Lastly, we analyzed the mechanisms of action of several existing and novel pharmacological treatments for PH. Lifelike elucidated the interplay between G-protein, IL6, activin, and BMP signaling. Those pathways regulate hallmark pathophysiological processes of PH, including vasoconstriction, endothelial barrier function, cell proliferation, and apoptosis. Discussion: The results highlight the importance of SRC, ERK1, AKT, and MLC activity in PH. The molecular pathways affected by existing and novel treatments for PH also converge on these molecules. Importantly, sotatercept affects SRC, ERK1, AKT, and MLC simultaneously. The present study shows the power of mining knowledge graphs using Lifelike's diverse set of data analytics functionalities for developing knowledge-driven hypotheses on PH pathophysiological and drug mechanisms and their interactions. We believe that Lifelike and our presented approach will be valuable for future mechanistic studies of PH, other diseases, and drugs.

Activin A, a Novel Chemokine, Induces Mouse NK Cell Migration via AKT and Calcium Signaling.

Natural killer (NK) cells can migrate quickly to the tumor site to exert cytotoxic effects on tumors, and some chemokines, including CXCL8, CXCL10 or and CXCL12, can regulate the migration of NK cells. Activin A, a member of the transforming growth factor ß (TGF-ß) superfamily, is highly expressed in tumor tissues and involved in tumor development and immune cell activation. In this study, we focus on the effects of activin A on NK cell migration. In vitro, activin A induced NK cell migration and invasion, promoted cell polarization and inhibited cell adhesion. Moreover, activin A increased Ca2+, p-SMAD3 and p-AKT levels in NK cells. An AKT inhibitor and Ca2+ chelator partially blocked activin A-induced NK cell migration. In vivo, exogenous activin A increased tumor-infiltrating NK cells in NS-1 cell solid tumors and inhibited tumor growth, and blocking endogenous activin A with anti-activin A antibody reduced tumor-infiltrating NK cells in 4T-1 cell solid tumors. These results suggest that activin A induces NK cell migration through AKT signaling and calcium signaling and may enhance the antitumor effect of NK cells by increasing tumor-infiltrating NK cells.

Activin A: a marker of mineral bone disorder in children with chronic kidney disease?

BACKGROUND: Activin A has been shown to enhance osteoclast activity and its inhibition results in bone growth. The potential role of activin A as a marker of chronic kidney disease-mineral bone disease (CKD-MBD) and its relationship with other markers has not been studied in children with CKD. METHODS: A cross sectional study was conducted among 40 children aged 2 to 18 years with CKD (Stage 2 to 5; 10 in each stage) and 40 matched controls. Activin A, cathepsin K, FGF-23, PTH, serum calcium, phosphorous and alkaline phosphatase in both groups were measured and compared. The correlation of activin A and markers of CKD-MBD was studied. A p value of < 0.05 was considered significant. RESULTS: The mean age of children with CKD was 9.30 ± 3.64 years. Mean levels of activin A in cases were 485.55 pg/ml compared to 76.19 pg/ml in controls (p < 0.001). FGF-23 levels in cases were 133.18 pg/ml while in controls it was 6.93 pg/ml (p < 0.001). Mean levels of cathepsin K were also significantly higher in cases as compared to controls. There was a progressive increase in activin A and cathepsin K levels with increasing stage of CKD. Activin A had a significant positive correlation with serum creatinine (r = 0.51; p < 0.001). CONCLUSIONS: Activin A levels progressively rise with advancing CKD stage. These findings suggest that activin A can be a potential early marker of CKD-MBD in children.

Design and synthesis of novel thiazole-derivatives as potent ALK5 inhibitors.

TGF-ß is an immunosuppressive cytokine and plays a key role in progression of cancer by inducing immunosuppression in tumor microenvironment. Therefore, inhibition of TGF-ß signaling pathway may provide a potential therapeutic intervention in treating cancers. Herein, we report the discovery of a series of novel thiazole derivatives as potent inhibitors of ALK5, a serine-threonine kinase which is responsible for TGF-ß signal transduction. Compound 29b was identified as a potent inhibitor of ALK5 with an IC50 value of 3.7 nM with an excellent kinase selectivity.

Gene Function is a Driver of Activin Signaling Pathway Evolution Following Whole-Genome Duplication in Rainbow Trout (Oncorhynchus mykiss).

The genomes of plant and animal species are influenced by ancestral whole-genome duplication (WGD) events, which have profound impacts on the regulation and function of gene networks. To gain insight into the consequences of WGD events, we characterized the sequence conservation and expression patterns of ohnologs in the highly duplicated activin receptor signaling pathway in rainbow trout (RBT). The RBT activin receptor signaling pathway is defined by tissue-specific expression of inhibitors and ligands and broad expression of receptors and Co-Smad signaling molecules. Signaling pathway ligands exhibited shared expression, while inhibitors and Smad signaling molecules primarily express a single dominant ohnolog. Our findings suggest that gene function influences ohnolog evolution following duplication of the activin signaling pathway in RBT.

Function of alveolar macrophages in lung cancer microenvironment.

BACKGROUND: Cancer tissues contain a wide variety of immune cells that play critical roles in suppressing or promoting tumor progression. Macrophages are one of the most predominant populations in the tumor microenvironment and are composed of two classes: infiltrating macrophages from the bone marrow and tissue-resident macrophages (TRMs). This review aimed to outline the function of TRMs in the tumor microenvironment, focusing on lung cancer. REVIEW: Although the functions of infiltrating macrophages and tumor-associated macrophages have been intensively analyzed, a comprehensive understanding of TRM function in cancer is relatively insufficient because it differs depending on the tissue and organ. Alveolar macrophages (AMs), one of the most important TRMs in the lungs, are replenished in situ, independent of hematopoietic stem cells in the bone marrow, and are abundant in lung cancer tissue. Recently, we reported that AMs support cancer cell proliferation and contribute to unfavorable outcomes. CONCLUSION: In this review, we introduce the functions of AMs in lung cancer and their underlying molecular mechanisms. A thorough understanding of the functions of AMs in lung cancer will lead to improved treatment outcomes.

The activin/BMP-2 chimera AB204 promotes periodontal tissue regeneration in a buccal dehiscence model: a pilot study.

PURPOSE: A combination of activin and bone morphogenetic protein-2 (BMP-2), termed AB204, has been shown to improve osteogenic potential with fewer side effects than BMP-2 alone. This study was performed to evaluate the effect of AB204 on periodontal tissue regeneration in a dog buccal dehiscence model. METHODS: Buccal dehiscence defects were created on the maxillary premolars (P1, P2, and P3) of 6 mongrel dogs. After 5 weeks, the dogs were randomly assigned to 1 of 3 groups: the control, collagen matrix (CM), and CM/AB204 groups. Grafting procedures were then performed. The dogs were sacrificed 8 weeks after the grafting procedure, and volumetric and histological analyses were conducted. RESULTS: The thickness of the buccal gingiva in the CM/AB204 group was greater than those in the other groups at 2 weeks (P<0.05). The ridge width in the AB204/CM group exceeded the width in the other groups at 4 and 8 weeks; however, the difference was not statistically significant. Histological analysis revealed that the CM/AB204 group demonstrated the formation of new bone surrounded by newly formed periodontal ligament and cementum (P=0.035). CONCLUSIONS: The combined application of CM and AB204 shows promise in facilitating the regeneration of periodontal attachment, including the formation of new bone, cementum, and periodontal ligament.

Bone regeneration using activin A/BMP2 chimera (AB204) with collagen membrane in rats with calvarial defects.

PURPOSE: Collagen has long been recognized as an excellent carrier for growth factors, and membrane-type collagen has been widely applied in dentistry for guided bone regeneration. This study was conducted to examine the effects of an activin A/BMP2 chimera (AB204) combined with a collagen membrane (CM) on bone repair in a rat calvarial defect model. METHODS: A unilateral calvarial defect measuring 5.0 mm was surgically created in 32 Sprague-Dawley rats. The rats were then randomly assigned to 1 of 4 groups, each consisting of 8 animals: control (untreated), CM (treated with a CM only), CM/bone morphogenetic protein 2 (BMP2) (treated with a CM and 1.0 µg of BMP2), and CM/AB204 (treated with a CM and 1.0 µg of AB204). Bone regeneration was evaluated using micro-computed tomography (CT) and histological analysis at 2 and 4 weeks following surgery. RESULTS: Micro-CT analysis revealed that bone formation in the CM/BMP2 and CM/AB204 groups was superior to that observed in the control and CM groups at both 2 and 4 weeks postoperatively. BMP2 induced greater bone regeneration than AB204 at 2 weeks; however, AB204 resulted in a greater bone volume at 4 weeks, achieving the highest values recorded. No significant differences were found between the CM/BMP2 and CM/AB204 groups at either time point (P>0.05). On histological examination, new bone formation was evident in both CM/BMP2 and CM/AB204 groups. CONCLUSIONS: Within the limitations of this study, the findings indicate that AB204 may enhance osteogenic potential when used in combination with CM for bone regeneration.

SMAD2/3-SMYD2 and developmental transcription factors cooperate with cell cycle inhibitors to guide tissue formation.

Tissue formation and organ homeostasis is achieved by precise coordination of proliferation and differentiation of stem cells and progenitors. While deregulation of these processes can result in degenerative disease or cancer, their molecular interplays remain unclear. Here we show that the switch of human pluripotent stem cell (hPSC) self-renewal to differentiation is associated with the induction of distinct cyclin dependent kinase inhibitors (CDKIs). In hPSCs, Activin/Nodal/TGFß signalling maintains CDKIs in a poised state via SMAD2/3-NANOG-OCT4-EZH2-SNON transcriptional complex. Upon gradual differentiation, CDKIs are induced by successive transcriptional complexes between SMAD2/3-SMYD2 and developmental regulators such as EOMES, thereby lengthening the G1 phase. This, in turn, induces SMAD2/3 transcriptional activity by blocking its linker phosphorylation. Such SMAD2/3-CDKI positive feedback loops drive the exit from pluripotency and stepwise cell fate specification that could be harnessed for producing cells for therapeutic applications. Our study uncovers fundamental mechanisms how cell fate specification is interconnected to cell cycle dynamics and provides insight to autonomous circuitries governing tissue self-formation.

Monocytes expressing activin A and CCR2 exacerbate chronic testicular inflammation by promoting immune cell infiltration.

STUDY QUESTION: Does the chemokine/chemokine receptor axis, involved in immune cell trafficking, contribute to the pathology of testicular inflammation and how does activin A modulate this network? SUMMARY ANSWER: Testicular chemokines and their receptors (especially those essential for trafficking of monocytes) are elevated in orchitis, and activin A modulates the expression of the chemokine/chemokine receptor network to promote monocyte/macrophage and T cell infiltration into the testes, causing extensive tissue damage. WHAT IS KNOWN ALREADY: The levels of CC motif chemokine receptor (CCR)2 and its ligand CC motif chemokine ligand (CCL)2 are increased in experimental autoimmune orchitis (EAO) compared with healthy testes, and mice deficient in CCR2 are protected from EAO-induced tissue damage. Activin A induces CCR2 expression in macrophages, promoting their migration. Moreover, there is a positive correlation between testicular activin A concentration and the severity of autoimmune orchitis. Inhibition of activin A activity by overexpression of follistatin (FST) reduces EAO-induced testicular damage. STUDY DESIGN, SIZE, DURATION: EAO was induced in 10-12-week-old male C57BL/6J (wild-type; WT) and B6.129P2-Ccr2tm1Mae/tm1Mae (Ccr2-/-) mice (n = 6). Adjuvant (n = 6) and untreated (n = 6) age-matched control mice were also included. Testes were collected at 50 days after the first immunization with testicular homogenate in complete Freund's adjuvant. In another experimental setup, WT mice were injected with a non-replicative recombinant adeno-associated viral vector carrying a FST315-expressing gene cassette (rAAV-FST315; n = 7-9) or an empty control vector (n = 5) 30 days prior to EAO induction. Appropriate adjuvant (n = 4-5) and untreated (n = 4-6) controls were also examined. Furthermore, human testicular biopsies exhibiting focal leukocytic infiltration and impaired spermatogenesis (n = 17) were investigated. Biopsies showing intact spermatogenesis were included as controls (n = 9). Bone-marrow-derived macrophages (BMDMs) generated from WT mice were treated with activin A (50 ng/ml) for 6 days. Activin-A-treated or untreated BMDMs were then co-cultured with purified mouse splenic T cells for two days to assess chemokine and cytokine production. PARTICIPANTS/MATERIALS, SETTING, METHODS: Quantitative real-time PCR (qRT-PCR) was used to analyze the expression of chemokines in total testicular RNA collected from mice. Immunofluorescence staining was used to detect activin A, F4/80, and CD3 expression in mouse testes. The expression of chemokine/chemokine-receptor-encoding genes was examined in human testicular biopsies by qRT-PCR. Correlations between chemokine expression levels and either the immune cell infiltration density or the mean spermatogenesis score were analyzed. Immunofluorescence staining was used to evaluate the expression of CD68 and CCR2 in human testicular biopsies. RNA isolated from murine BMDMs was used to characterize these cells in terms of their chemokine/chemokine receptor expression levels. Conditioned media from co-cultures of BMDMs and T cells were collected to determine chemokine levels and the production of pro-inflammatory cytokines tumor necrosis factor (TNF) and interferon (IFN)-γ by T cells. MAIN RESULTS AND THE ROLE OF CHANCE: Induction of EAO in the testes of WT mice increased the expression of chemokine receptors such as Ccr1 (P < 0.001), Ccr2 (P < 0.0001), Ccr3 (P < 0.0001), Ccr5 (P < 0.0001), CXC motif chemokine receptor (Cxcr)3 (P < 0.01), and CX3C motif chemokine receptor (Cx3cr)1 (P < 0.001), as well as that of most of their ligands. Ccr2 deficiency reversed some of the changes associated with EAO by reducing the expression of Ccr1 (P < 0.0001), Ccr3 (P < 0.0001), Ccr5 (P < 0.01), Cxcr3 (P < 0.001), and Cx3cr1 (P < 0.0001). Importantly, the biopsies showing impaired spermatogenesis and concomitant focal leukocytic infiltration exhibited higher expression of CCL2 (P < 0.01), CCR1 (P < 0.05), CCR2 (P < 0.001), and CCR5 (P < 0.001) than control biopsies with no signs of inflammation and intact spermatogenesis. The gene expression of CCR2 and its ligand CCL2 correlated positively with the immune cell infiltration density (P < 0.05) and negatively with the mean spermatogenesis score (P < 0.001). Moreover, CD68+ macrophages expressing CCR2 were present in human testes with leukocytic infiltration with evidence of tubular damage. Treatment of BMDMs, as surrogates for testicular macrophages, with activin A increased their expression of Ccr1, Ccr2, and Ccr5 while reducing their expression of Ccl2, Ccl3, Ccl4, Ccl6, Ccl7 Ccl8, and Ccl12. These findings were validated in vivo, by showing that inhibiting activin A activity by overexpressing FST in EAO mice decreased the expression of Ccr2 (P < 0.05) and Ccr5 (P < 0.001) in the testes. Interestingly, co-culturing activin-A-treated BMDMs and T cells reduced the levels of CCL2 (P < 0.05), CCL3/4 (P < 0.01), and CCL12 (P < 0.05) in the medium and attenuated the production of TNF (P < 0.05) by T cells. The majority of cells secreting activin A in EAO testes were identified as macrophages. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: BMDMs were used as surrogates for testicular macrophages. Hence, results obtained from the in vitro experiments might not be fully representative of the situation in the testes in vivo. Moreover, since total RNA was extracted from the testicular tissue to examine chemokine expression, the contributions of individual cell types as producers of specific chemokines may have been overlooked. WIDER IMPLICATIONS OF THE FINDINGS: Our data indicate that macrophages are implicated in the development and progression of testicular inflammation by expressing CCR2 and activin A, which ultimately remodel the chemokine/chemokine receptor network and recruit other immune cells to the site of inflammation. Consequently, inhibition of CCR2 or activin A could serve as a potential therapeutic strategy for reducing testicular inflammation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the International Research Training Group in 'Molecular pathogenesis on male reproductive disorders', a collaboration between Justus Liebig University (Giessen) and Monash University (Melbourne) (GRK1871/1-2) funded by the Deutsche Forschungsgemeinschaft and Monash University, a National Health and Medical Research Council of Australia Ideas Grant (1184867), and the Victorian Government's Operational Infrastructure Support Programme. The authors declare no competing financial interests.

Inhibition of activin receptor 2 signalling ameliorates metabolic dysfunction-associated steatotic liver disease in western diet/L-NAME induced cardiometabolic disease.

OBJECTIVE: Blockade of activin 2 receptor (ACVR2) signaling has been shown to improve insulin sensitivity and aid in weight loss. Inhibition of ACVR2 signaling restores cardiac function in multiple heart failure models. However, its potential in the treatment of obesity-related cardiometabolic disease remains unknown. Here, we investigated targeting ACVR2 signaling in cardiometabolic disease manifested with metabolic dysfunction-associated steatotic liver disease (MASLD). METHODS: Mice were fed a high-fat, high-sugar diet combined with the administration of nitric oxide synthase inhibitor L-NAME in drinking water, which causes hypertensive stress. For the last eight weeks, the mice were treated with the soluble ACVR2B decoy receptor (sACVR2B-Fc). RESULTS: sACVR2B-Fc protected against the development of comorbidities associated with cardiometabolic disease. This was most pronounced in the liver where ACVR2 blockade attenuated the development of MASLD including cessation of pro-fibrotic activation. It also significantly reduced total plasma cholesterol levels, impeded brown adipose tissue whitening, and improved cardiac diastolic function. In vitro, ACVR2 ligands activin A, activin B and GDF11 induced profibrotic signaling and the proliferation of human cardiac fibroblasts. CONCLUSIONS: Blockade of ACVR2B exerts broad beneficial effects for therapy of cardiometabolic disease. By reducing obesity, ameliorating cardiovascular deterioration and restraining MASLD, blockade of ACVR2B signaling proves a potential target in MASLD and its comorbidities.

Anemia in patients receiving anticancer treatments: focus on novel therapeutic approaches.

Anemia is common in cancer patients and impacts on quality of life and prognosis. It is typically multifactorial, often involving different pathophysiological mechanisms, making treatment a difficult task. In patients undergoing active anticancer treatments like chemotherapy, decreased red blood cell (RBC) production due to myelosuppression generally predominates, but absolute or functional iron deficiency frequently coexists. Current treatments for chemotherapy-related anemia include blood transfusions, erythropoiesis-stimulating agents, and iron supplementation. Each option has limitations, and there is an urgent need for novel approaches. After decades of relative immobilism, several promising anti-anemic drugs are now entering the clinical scenario. Emerging novel classes of anti-anemic drugs recently introduced or in development for other types of anemia include activin receptor ligand traps, hypoxia-inducible factor-prolyl hydroxylase inhibitors, and hepcidin antagonists. Here, we discuss their possible role in the treatment of anemia observed in patients receiving anticancer therapies.

BRCC3 Regulation of ALK2 in Vascular Smooth Muscle Cells: Implication in Pulmonary Hypertension.

BACKGROUND: An imbalance of antiproliferative BMP (bone morphogenetic protein) signaling and proliferative TGF-ß (transforming growth factor-ß) signaling is implicated in the development of pulmonary arterial hypertension (PAH). The posttranslational modification (eg, phosphorylation and ubiquitination) of TGF-ß family receptors, including BMPR2 (bone morphogenetic protein type 2 receptor)/ALK2 (activin receptor-like kinase-2) and TGF-ßR2/R1, and receptor-regulated Smads significantly affects their activity and thus regulates the target cell fate. BRCC3 modifies the activity and stability of its substrate proteins through K63-dependent deubiquitination. By modulating the posttranslational modifications of the BMP/TGF-ß-PPARγ pathway, BRCC3 may play a role in pulmonary vascular remodeling, hence the pathogenesis of PAH. METHODS: Bioinformatic analyses were used to explore the mechanism by which BRCC3 deubiquitinates ALK2. Cultured pulmonary artery smooth muscle cells (PASMCs), mouse models, and specimens from patients with idiopathic PAH were used to investigate the rebalance between BMP and TGF-ß signaling in regulating ALK2 phosphorylation and ubiquitination in the context of pulmonary hypertension. RESULTS: BRCC3 was significantly downregulated in PASMCs from patients with PAH and animals with experimental pulmonary hypertension. BRCC3, by de-ubiquitinating ALK2 at Lys-472 and Lys-475, activated receptor-regulated Smad1/5/9, which resulted in transcriptional activation of BMP-regulated PPARγ, p53, and Id1. Overexpression of BRCC3 also attenuated TGF-ß signaling by downregulating TGF-ß expression and inhibiting phosphorylation of Smad3. Experiments in vitro indicated that overexpression of BRCC3 or the de-ubiquitin-mimetic ALK2-K472/475R attenuated PASMC proliferation and migration and enhanced PASMC apoptosis. In SM22α-BRCC3-Tg mice, pulmonary hypertension was ameliorated because of activation of the ALK2-Smad1/5-PPARγ axis in PASMCs. In contrast, Brcc3-/- mice showed increased susceptibility of experimental pulmonary hypertension because of inhibition of the ALK2-Smad1/5 signaling. CONCLUSIONS: These results suggest a pivotal role of BRCC3 in sustaining pulmonary vascular homeostasis by maintaining the integrity of the BMP signaling (ie, the ALK2-Smad1/5-PPARγ axis) while suppressing TGF-ß signaling in PASMCs. Such rebalance of BMP/TGF-ß pathways is translationally important for PAH alleviation.