Protective effects of Baicalin injection on severe acute pancreatitis through regulating follistatin-like-1 signaling pathway by down-regulating miR-429 expression in mice

Abstract In China, Scutellaria is used for treating inflammatory-related diseases. Baicalin is the main active component of Scutellaria and has protective effects on acute pancreatitis. However, the mechanism of Baicalin is still unclear. In this study, the protective effects of baicalin on acute pancreatitis induced by taurocholate and its mechanism are investigated. In this study, mice were randomly divided into three groups: sham operation, model, and treatment groups. Acute pancreatitis in mice was induced by intraperitoneal injection of taurocholate (35 mg/kg). The treatment group was given baicalin (100 mg/kg) 2 h before acute pancreatitis induction. The mRNA expression levels of miR-429, nuclear factor kappa B65(NF-kB65), toll-like receptor 4(TLR4), TNF receptor associated factor6 (TRAF6), NF-kappa-B inhibitor(IkB), Follistatin-like 1 (FSTL1), and interleukin-1 receptor-associated kinase (IRAK) in the liver tissues 24 h after intraperitoneal injection were detected by RT-PCR. Then, the expression levels of NF-kB65, p-NF-κB65, TLR4, TRAF6, IkB, FSTL1, IRAK, p- IRAK, and p- IkB-а proteins were detected by Western blot. IL-6, TNF-α and IL-1 ß in plasma were measured by ELISA, and histopathological changes in the pancreases of the mice were observed. The results showed that after baicalin treatment, miR-429 expression in the pancreatic tissues and the expression levels of NF-kB65, TLR4, TRAF6, p-IkB-а, FSTL1, and p-IRAK decreased. Similarly, pancreatic myeloperoxidase (MPO) activity and the plasma levels of IL-6, TNF-а, IL-12, IL-1ß1, endotoxin, serum amylase, and lipase were reduced. Thus, the pancreatic injury induced by taurocholate was alleviated. The present study indicates that pretreatment with Baicalin can alleviate acute pancreatic injury induced by taurocholate in mice. The mechanism may be associated with the decreased miR-429 expression, reduced FSTL1 signaling pathway activity, TLR4 and TLR4/MyD88 signaling pathway inhibition, and reduced pancreatic inflammation. FSTL1 is the regulatory target for miR-429

Detection of follistatin-based inhibitors of the TGF-ß signaling pathways in serum/plasma by means of LC-HRMS/MS and Western blotting.

Cytokines of the transforming growth factor beta (TGF-ß) superfamily such as myostatin and activin A are considered as key regulators of skeletal muscle mass. In vivo, their activity is controlled by different binding proteins such as follistatin (FST), whose interaction with the circulating growth factors prevents activation of the activin type II receptors. FST-based protein therapeutics are therefore not only promising drug candidates for the treatment of muscular diseases but also potential performance-enhancing agents in sports. Within this study, two complementary detection assays for FST-based inhibitors of the TGF-ß signaling pathways in doping control serum and plasma samples were developed by using both monomeric FST and dimeric FST-Fc fusion proteins as model compounds. The initial testing procedure is based on immunoaffinity purification, tryptic digestion, and LC-HRMS/MS, offering high specificity by targeting tryptic signature peptides of FST. As the glycoprotein is also produced endogenously, the confirmation method employs immunoaffinity purification, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and Western blotting in order to detect the intact proteins and differentiate synthetic FST-Fc constructs from naturally occurring FST isoforms. Both assays were found to be highly specific with an estimated detection limit of 10 ng/ml. Moreover, a commercial sandwich enzyme-linked immunosorbent assay was used to determine endogenous FST values. The detected FST serum levels of healthy volunteers were found below 5 ng/ml, which is in accordance with reference values from the literature and below the doping control detection methods' limit of detection (LOD). The presented assays expand the range of available tests for emerging doping agents, and the initial testing procedure can readily be modified to include further protein drugs.

Leveraging Quantitative Systems Pharmacology Approach into Development of Human Recombinant Follistatin Fusion Protein for Duchenne Muscular Dystrophy.

Quantitative understanding about the dynamics of drug-target interactions in biological systems is essential, especially in rare disease programs with small patient populations. Follistatin, by antagonism of myostatin and activin, which are negative regulators of skeletal muscle and inflammatory response, is a promising therapeutic target for Duchenne Muscular Dystrophy. In this study, we constructed a quantitative systems pharmacology model for FS-EEE-Fc, a follistatin recombinant protein to investigate its efficacy from dual target binding, and, subsequently, to project its human efficacious dose. Based on model simulations, with an assumed efficacy threshold of 7-10% muscle volume increase, 3-5 mg/kg weekly dosing of FS-EEE-Fc is predicted to achieve meaningful clinical outcome. In conclusion, the study demonstrated an application of mechanism driven approach at early stage of a rare disease drug development to support lead compound optimization, enable human dose, pharmacokinetics, and efficacy predictions.

Intravascular Follistatin gene delivery improves glycemic control in a mouse model of type 2 diabetes.

Type 2 diabetes (T2D) manifests from inadequate glucose control due to insulin resistance, hypoinsulinemia, and deteriorating pancreatic ß-cell function. The pro-inflammatory factor Activin has been implicated as a positive correlate of severity in T2D patients, and as a negative regulator of glucose uptake by skeletal muscle, and of pancreatic ß-cell phenotype in mice. Accordingly, we sought to determine whether intervention with the Activin antagonist Follistatin can ameliorate the diabetic pathology. Here, we report that an intravenous Follistatin gene delivery intervention with tropism for striated muscle reduced the serum concentrations of Activin B and improved glycemic control in the db/db mouse model of T2D. Treatment reversed the hyperglycemic progression with a corresponding reduction in the percentage of glycated-hemoglobin to levels similar to lean, healthy mice. Follistatin gene delivery promoted insulinemia and abundance of insulin-positive pancreatic ß-cells, even when treatment was administered to mice with advanced diabetes, supporting a mechanism for improved glycemic control associated with maintenance of functional ß-cells. Our data demonstrate that single-dose intravascular Follistatin gene delivery can ameliorate the diabetic progression and improve prognostic markers of disease. These findings are consistent with other observations of Activin-mediated mechanisms exerting deleterious effects in models of obesity and diabetes, and suggest that interventions that attenuate Activin signaling could help further understanding of T2D and the development of novel T2D therapeutics.

Follistatin-288-Fc Fusion Protein Promotes Localized Growth of Skeletal Muscle.

Follistatin is an endogenous glycoprotein that promotes growth and repair of skeletal muscle by sequestering inhibitory ligands of the transforming growth factor-ß superfamily and may therefore have therapeutic potential for neuromuscular diseases. Here, we sought to determine the suitability of a newly engineered follistatin fusion protein (FST288-Fc) to promote localized, rather than systemic, growth of skeletal muscle by capitalizing on the intrinsic heparin-binding ability of the follistatin-288 isoform. As determined by surface plasmon resonance and cell-based assays, FST288-Fc binds to activin A, activin B, myostatin (growth differentiation factor GDF8), and GDF11 with high affinity and neutralizes their activity in vitro. Intramuscular administration of FST288-Fc in mice induced robust, dose-dependent growth of the targeted muscle but not of surrounding or contralateral muscles, in contrast to the systemic effects of a locally administered fusion protein incorporating activin receptor type IIB (ActRIIB-Fc). Furthermore, systemic administration of FST288-Fc in mice did not alter muscle mass or body composition as determined by NMR, which again contrasts with the pronounced systemic activity of ActRIIB-Fc when administered by the same route. Subsequent analysis revealed that FST288-Fc in the circulation undergoes rapid proteolysis, thereby restricting its activity to individual muscles targeted by intramuscular administration. These results indicate that FST288-Fc can produce localized growth of skeletal muscle in a targeted manner with reduced potential for undesirable systemic effects. Thus, FST288-Fc and similar agents may be beneficial in the treatment of disorders with muscle atrophy that is focal, asymmetric, or otherwise heterogeneous.

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

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

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.

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

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

Follistatin, an activin antagonist, ameliorates renal interstitial fibrosis in a rat model of unilateral ureteral obstruction.

Activin, a member of the TGF-ß superfamily, regulates cell growth and differentiation in various cell types. Activin A acts as a negative regulator of renal development as well as tubular regeneration after renal injury. However, it remains unknown whether activin A is involved in renal fibrosis. To clarify this issue, we utilized a rat model of unilateral ureteral obstruction (UUO). The expression of activin A was significantly increased in the UUO kidneys compared to that in contralateral kidneys. Activin A was detected in glomerular mesangial cells and interstitial fibroblasts in normal kidneys. In UUO kidneys, activin A was abundantly expressed by interstitial α-SMA-positive myofibroblasts. Administration of recombinant follistatin, an activin antagonist, reduced the fibrotic area in the UUO kidneys. The number of proliferating cells in the interstitium, but not in the tubules, was significantly lower in the follistatin-treated kidneys. Expression of α-SMA, deposition of type I collagen and fibronectin, and CD68-positive macrophage infiltration were significantly suppressed in the follistatin-treated kidneys. These data suggest that activin A produced by interstitial fibroblasts acts as a potent profibrotic factor during renal fibrosis. Blockade of activin A action may be a novel approach for the prevention of renal fibrosis progression.

Systemic administration of follistatin288 increases muscle mass and reduces fat accumulation in mice.

The present study describes the physiological response associated with daily subcutaneous injection of mice with recombinant follistatin288. This systemic administration of follistatin288 increases the follistatin levels in serum, indicating that the protein enters the circulation. The data suggest that a dose-dependent increase in body lean mass also occurs, together with an increase in muscle mass, possibly as a result of an increase in the size of the muscle fibers. After thirteen weeks of treatment, metabolic changes were observed; additionally, the switching of muscle fiber types was also apparent through myosin heavy chain remodeling, implying that changes are occurring at the molecular level. Furthermore, an increase in the muscle mass was associated with a significant decrease in the body fat mass. Overall, this study raises the possibility for the use of follistatin288 as an agent to treat muscle wasting diseases and/or to restrict fat accumulation by systemic administration of the protein.

Uso tópico de 5-azacitidina melhora a cicatrização de feridas cutâneas de roedores por meio do sistema ativina/folistatina / Topical 5-azacytidine accelerates skin wound healing in rats

O desenvolvimento de métodos que tem por objetivo acelerar e melhorar a qualidade do processo de cicatrização de feridas tem impacto positivo na condução de distúrbios de cicatrização associados a inúmeras condições médicas. Neste estudo, avaliamos os efeitos moleculares, celulares e clínicos da aplicação tópica de 5-azacitidina na cicatrização de feridas em ratos. De acordo com estudos pregressos, a 5-azacitidina reduz a expressão de folistatina, que é um regulador negativo das ativinas. Estas, por sua vez, promovem o crescimento de células em diferentes tecidos, incluindo a pele. Ratos Wistar machos com oito semanas de vida foram submetidos a um ferimento cutâneo com punch de oito milímetros na região dorsal. A seguir os ratos foram aleatoriamente separados em grupo controle (veículo) ou submetidos à aplicação tópica de 5-azacitidina (10 mM), uma vez por dia por até 12 dias, iniciando-se no terceiro dia após a lesão. A documentação fotográfica e coleta de amostras ocorreram nos dias 5, 9 e 15. O emprego desta droga resultou em aceleração da cicatrização da ferida, (99,7±7,0% versus 71,2±2,8% no dia 15, p <0,01). Este resultado clínico foi acompanhado pela redução de aproximadamente três vezes na expressão protéica de folistatina. O exame histológico da pele revelou re-epitelização eficiente com aumento da expressão de queratinócitos e aumento significativo na expressão do gene de TGF-β além da diminuição significativa de citocinas, tais como TNF-α e IL-10. Analisamos também a proliferação celular na lesão de pele através do método de incorporação de BrdU. O número de células positivas para BrdU aumentou significativamente quando comparado ao controle. No entanto, quando folistatina exógena foi aplicada na pele em paralelo ao tratamento tópico de 5-azacitidina a maioria dos benefícios do medicamento foi perdida.

The development of new methods aimed at improving wound healing may have an impact on the outcomes of a number of medical conditions. Here we evaluate the molecular and clinical effects of topical 5-azacytidine, a compound used in myelodysplasia, on the wound healing in rats. According to previous studies, 5-Azacytidine decreases the expression of follistatin 1, which is a negative regulator of activins. Activins, in turn, promote cell growth in different tissues, including the skin. Eight-week old male Wistar rats were submitted to an 8 mm punchwound in the dorsal region. After three days, rats were randomly assigned to either control or topical application of a solution containing 5-azacytidine (10mM), once a day. Photo documentation and collection of samples occurred at days 5, 9 and 15. Overall, 5-azacytidine resulted on a significant acceleration of complete wound healing (99.7% ±0.7.0 vs. 71.2%±2.8 on days 15; n=10; p<0,01). This was accompanied by an up to 3-fold reduction in follistatin expression. Histological examination of the skin revealed efficient reepithelization with increase in gene expression of TGF-β and keratinocytes markers, involucrin and citokeratin, besides the significant decrease of cytokines such as TNF-α and IL-10. In addition, we analyzed cell proliferation in injured skin employing the BrdU incorporation method. The treatment with 5-azacytidine led to a progressive increase of BrdU positive cells. Finally when recombinant follistatin was employed in the skin in parallel to topical 5-azacytidine most of the benefits of the drug were lost. Thus, 5-azacytidine acts, at least in part, through the follistatin/activin pathway to improve wound healing in rats. This study belongs to online research process Caring in Nursing and Health.

Injection of duck recombinant follistatin fusion protein into duck muscle tissues stimulates satellite cell proliferation and muscle fiber hypertrophy.

Follistatin (FST) can inhibit the expression of myostatin, which is a predominant inhibitor of muscle development. The potential application of myostatin-based technology has been prompted in different ways in agriculture. We previously constructed an expression vector of duck FST and isolated the FST fusion protein. After the protein was purified and refolded, it was added to the medium of duck myoblasts cultured in vitro. The results show that the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide value of the myoblasts in the duck FST treatment group is higher than that in the control group, which indicates that the duck FST fusion protein exhibits the biological activities that can accelerate myoblast proliferation. To further investigate the roles of duck FST on muscle development, we injected the protein into the duck muscle tissues in vivo. The results show that both the duck muscle fiber cross-sectional area and the satellite cell activation frequency are influenced more in the FST treatment group than they are in the control group. In addition to these phenomena, expression of MyoD and Myf5 were increased, and the expression of myostatin was decreased. Together, these results suggest the potential for using duck FST fusion protein to inhibit myostatin activity and subsequently to enhance muscle growth in vivo. The mechanism by which FST regulates muscle development in the duck is similar to that in mammals and fishes.

Hair regrowth following a Wnt- and follistatin containing treatment: safety and efficacy in a first-in-man phase 1 clinical trial.

Research has shown the importance of follistatin, Wnt 7a, and wound healing growth factors on the stimulation of bulge cells and inter-follicular stem cells to induce hair growth. We have studied the effects of a bioengineered, non-recombinant, human cell-derived formulation, termed Hair Stimulating Complex (HSC), containing these factors to assess its hair growth activity in male pattern baldness. HSC showed in vitro Wnt activity and contained follistatin, KGF, and VEGF. The clinical study was a double-blind, placebo-controlled, randomized single site trial and was designed to evaluate safety of the HSC product and assess efficacy in stimulating hair growth. All 26 subjects tolerated the single, intradermal injection of HSC procedures well, and no signs of an adverse reaction were reported. Histopathological evaluation of the treatment site biopsies taken at 22 and 52 weeks post-treatment revealed no abnormal morphology, hamartomas, or other pathological responses. Trichoscan image analysis of HSC-treated sites at 12 and 52 weeks showed significant improvements in hair growth over the placebo. At the initial 12-week evaluation period, HSC-treated sites demonstrated an increase in hair shaft thickness (6.3%±2.5% vs. -0.63%±2.1%; P=0.046), thickness density (12.8%±4.5% vs. -0.2%±2.9%; P=0.028), and terminal hair density (20.6±4.9% vs. 4.4±4.9%; P=0.029). At one year, a statistically significant increase in total hair count (P=0.032) continued to be seen. These results demonstrate that a single intradermal administration of HSC improved hair growth in subjects with androgenetic alopecia and is a clinical substantiation of previous preclinical research with Wnts, follistatin, and other growth factors associated with wound healing and regeneration.

Upregulation of activin signaling in experimental colitis.

Several lines of studies have suggested that activins are critical mediators of inflammation and tissue repair. As activins and their receptors are expressed in the gastrointestinal tract, we tested the hypothesis that activin signaling is involved in the development of colitis by using two murine models of colitis induced by dextran sodium sulfate (DSS) or in mdr1a-/- mice. By immunohistochemistry, expression of activins was found increased in both models and correlated with the severity of inflammation. Activin expression was observed in macrophages as well as in some nonmacrophage cells. Furthermore, while activin receptors are normally expressed in colonic epithelial cells, their expression was further increased in both epithelial cells and inflammatory cells in inflamed colonic mucosa. Moreover, in vitro studies showed that activin A inhibited proliferation and induced apoptosis of intestinal epithelial cells, and this growth inhibition was largely reversed by administration of the activin inhibitor, follistatin. Because we also observed an increased number of apoptotic epithelial cells in both colitis models, the upregulation of activins occurring in colitis could be involved both in the inflammatory process and in growth inhibition of the intestinal epithelium. Importantly, in vivo administration of follistatin attenuated inflammatory cell infiltration during colitis. Rectal bleeding was reduced, and the integrity of epithelium was preserved in the DSS/follistatin-treated group compared with the group treated with DSS alone. Bromodeoxyuridine incorporation studies showed an increase in proliferative epithelial cells in the DSS/follistatin-treated group, suggesting that follistatin accelerates epithelial cell proliferation/repair during colitis. Overall, our results reveal that activin signaling may play an important role in the pathogenesis and resolution of colitis. These findings suggest new therapeutic options in inflammatory bowel diseases.

Delivery of recombinant follistatin lessens disease severity in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is the most common genetic cause of infant mortality. SMA is caused by loss of functional survival motor neuron 1 (SMN1), resulting in death of spinal motor neurons. Current therapeutic research focuses on modulating the expression of a partially functioning copy gene, SMN2, which is retained in SMA patients. However, a treatment strategy that improves the SMA phenotype by slowing or reversing the skeletal muscle atrophy may also be beneficial. Myostatin, a member of the TGF-beta super-family, is a potent negative regulator of skeletal muscle mass. Follistatin is a natural antagonist of myostatin, and over-expression of follistatin in mouse muscle leads to profound increases in skeletal muscle mass. To determine whether enhanced muscle mass impacts SMA, we administered recombinant follistatin to an SMA mouse model. Treated animals exhibited increased mass in several muscle groups, elevation in the number and cross-sectional area of ventral horn cells, gross motor function improvement and mean lifespan extension by 30%, by preventing some of the early deaths, when compared with control animals. SMN protein levels in spinal cord and muscle were unchanged in follistatin-treated SMA mice, suggesting that follistatin exerts its effect in an SMN-independent manner. Reversing muscle atrophy associated with SMA may represent an unexploited therapeutic target for the treatment of SMA.

Role of serum myostatin during the lactation period.

Myostatin, also known as GDF-8 (Growth/Differentiation Factor-8), is a member of the TGF-beta superfamily that negatively regulates skeletal muscle mass in mammals. Mutation of the myostatin gene in mice, cattle, and humans causes a massively developed skeletal muscle, characterized by muscle hypertrophy and hyperplasia. Although myostatin is predominantly expressed in skeletal muscle tissue, several recent studies have shown the presence of myostatin protein in blood and suggested a possible role for circulating myostatin in the regulation of skeletal muscle mass. In the present study, we examined changes in the levels of active form myostatin (13 kDa) in serum after birth by Western blot analysis to predict the role of serum myostatin in early postnatal muscle growth in the rat. Interestingly, the amount of active form myostatin in serum increased after birth and then decreased along with ageing after weaning. To clarify the role of increased serum myostatin during the postnatal period, we administrated follistatin, an inhibitor of myostatin activity, into postnatal rats intraperitoneally just after birth. Follistatin-administration during the postnatal period caused selective hypertrophy of type II muscle fibers in the soleus muscle. These results demonstrate that myostatin in serum acts on skeletal muscle and negatively regulates early postnatal muscle growth.

Possible endocrine control by follistatin 315 during liver regeneration based on changes in the activin receptor after a partial hepatectomy in rats.

BACKGROUND/AIMS: Activin A is an autocrine inhibitor of cell growth in the liver. The biological activity of activin A is mediated by a heteromeric receptor complex. Follistatin (FS) binds to activin and inhibits its biological effects, and acts as a negative regulator of muscle cells. The role of activin receptors during liver regeneration following a hepatectomy has not been fully assessed. This study investigates the mechanism underlying how activin receptors regulate hepatocyte growth, and the effects of intravenous administration of FS during liver regeneration. METHODOLOGY: The expression of both activins and activin receptors in the liver after a 70% partial hepatectomy (HT) was assessed by RT-PCR and immunohistochemistry. FS 315 or 288 was infused for different periods of time based on changes in hepatocyte activin receptor expression after HT. RESULTS: Activin receptor expression peaked between 48 and 72 hours after HT. 72 hours after HT, an injection of FS 315 resulted in a more potent stimulation of DNA synthesis and produced a greater increase in body weight compared with the control rats. CONCLUSIONS: The expression of activin receptors after peak DNA synthesis might be a key component in the downregulation of DNA synthesis. Intravenous administration of FS 315 might promote liver regeneration and have anabolic actions.

Follistatin allows efficient retroviral-mediated gene transfer into rat liver.

Retroviral vectors are widely used tools for gene therapy. However, in vivo gene transfer is only effective in dividing cells, which, in liver, requires a regenerative stimulus. Follistatin is effective in promoting liver regeneration after 90% and 70% hepatectomy in rats. We studied its efficacy on liver regeneration and retroviral-mediated gene delivery in 50% hepatectomized rats. When human recombinant follistatin was infused into the portal vein immediately after 50% hepatectomy, hepatocyte proliferation was significantly higher than in control 50% hepatectomized rats. A single injection of virus particles administered 23 h after follistatin infusion resulted in more than 20% gene transduction efficiency in hepatocytes compared to 3% in control rats. It is concluded that a single injection of follistatin induces onset of proliferation in 50% hepatectomized rats and allows efficient retroviral-mediated gene transfer to the liver.

Developmental profiles of activin betaA, betaB, and follistatin expression in the zebrafish ovary: evidence for their differential roles during sexual maturation and ovulatory cycle.

Our recent experiments showed that gonadotropin(s) stimulated activin betaA and follistatin expression through the cAMP-PKA pathway but suppressed betaB via a cAMP-dependent but PKA-independent pathway in cultured zebrafish follicle cells. Given that pituitary gonadotropins are the major hormones controlling the development and function of the ovary, the differential expression of activin betaA and betaB as well as follistatin in response to gonadotropin(s) raises an interesting question about the temporal expression patterns of these molecules in vivo during sexual maturation and ovulatory cycle. Three experiments were performed in the present study. In the first experiment using sexually immature zebrafish, we followed the expression of activin betaA, betaB, and follistatin at the whole ovary level during a 10-day period in which the ovary developed from the primary growth stage to the one with nearly full-grown follicles. Activin betaA expression was very low at the primary growth stage but significantly increased with the growth of the ovary, and its rise was accompanied by an increase in follistatin expression. In contrast, the expression of activin betaB could be easily detected in the ovary of all stages; however, it did not exhibit an obvious trend of variation during the development. The second experiment examined the stage-dependent expression of activin betaA, betaB, and follistatin at the follicle level in the adult mature zebrafish. The expression of activin betaA was again low in the follicles during the primary growth stage, but exhibited a phenomenal increase after the follicles entered vitellogenesis with the peak level reached at midvitellogenic stage; in contrast, activin betaB mRNA could be easily detected at all stages with a slight increase during follicle growth. The expression of follistatin, on the other hand, also increased significantly during vitellogenesis; however, its level dropped sharply after reaching the peak at the midvitellogenic stage. In the third experiment, we investigated the dynamic changes of the ovarian activin betaA, betaB, and follistatin expression during the daily ovulatory cycle. The expression of activin betaA and follistatin gradually increased from 1800 h onward and reached the peak level around 0400 h when the germinal vesicles had migrated to the periphery in the full-grown oocytes. In contrast, activin betaB expression steadily declined, although not statistically significant, during the same period, but increased sharply at 0700 h when mature oocytes started to appear in most of the ovaries collected. In conclusion, activin betaA and betaB exhibit distinct expression patterns during the development of the ovary and the daily ovarian cycle of the zebrafish. It seems that activin betaA is involved in promoting ovary and follicle growth, whereas activin betaB may have a tonic role throughout follicle development but becomes critical at the late stage of oocyte maturation and/or ovulation.

Role of activin A as a mediator of in vitro endometrial stromal cell decidualization via the cyclic adenosine monophosphate pathway.

OBJECTIVE: To elucidate the regulation and role of activin A in endometrial stromal decidualization. DESIGN: In vitro model of human stromal cell decidualization with cyclic adenosine monophosphate (cAMP) used to evaluate expression of activin A and to evaluate the effect of the addition of follistatin, a known activin inhibitor, on expression of the decidualized phenotype (as measured by levels of insulin-like growth factor binding protein-1 [IGFBP-1]). SETTING: Academic research environment. PATIENT(S): Four premenopausal, normally cycling subjects (age range: 32-40 years). INTERVENTION(S): Endometrial samples were obtained from the subjects after informed consent was obtained. Endometrial stromal cells were treated with cAMP (decidualizing stimulus) and 50 ng/mL, 100 ng/mL, and 200 ng/mL of follistatin for 48 hours. MAIN OUTCOME MEASURE(S): Levels of IGFBP-1 secreted from cells decidualized in the absence and presence of three different concentrations of follistatin. RESULT(S): Addition of follistatin, a known binding protein inhibitor of activin A, resulted in a dose-dependent inhibition of IGFBP-1 secreted into conditioned medium, with the greatest decrease observed at 4 days of decidualization. Cells treated with cAMP and 50 ng/mL, 100 ng/mL, and 200 ng/mL of follistatin demonstrated 67.3%, 58.6%, and 35.5%, respectively, of the IGFBP-1 levels observed with cAMP but without follistatin. CONCLUSION(S): These data suggest that activin A is a necessary component of the cAMP pathway leading to endometrial stromal decidualization. The role of activin A in regulating endometrial stromal decidualization and its known promotion of the invasive phenotype of the trophoblast suggest unique autocrine and paracrine interactions at the maternal/fetal interface during implantation, which might have important clinical implications for the understanding and treatment of fertility and pregnancy disorders.