Inter- and intra-session variability of compression strain gauge for the adductor groin squeeze test on soccer athletes.

The importance of hip adductor strength for injury prevention and performance benefits is well documented. The purpose of this study was to establish the intra- and inter-day variability of peak force (PF) of a groin squeeze protocol using a custom-designed compression strain gauge device. Sixteen semi-professional soccer players completed three trials over three separate testing occasions with at least 24-h rest between each session. The main findings were that the compression strain gauge was a reliable device for measuring PF within and between days. All intraclass correlations were higher than 0.80 and coefficients of variations were below 10% across the different sessions and trials. Due to the information gained through the compression strain gauge, the higher sampling frequency utilized, portability, and the relatively affordable price, this device offers an effective alternative for measuring maximal strength for hip adduction.

Oocyte and cumulus cell cooperativity and metabolic plasticity under the direction of oocyte paracrine factors.

Mammalian oocytes develop and mature in a mutually dependent relationship with surrounding cumulus cells. The oocyte actively regulates cumulus cell differentiation and function by secreting soluble paracrine oocyte-secreted factors (OSFs). We characterized the molecular mechanisms by which two model OSFs, cumulin and BMP15, regulate oocyte maturation and cumulus-oocyte cooperativity. Exposure to these OSFs during mouse oocyte maturation in vitro altered the proteomic and multispectral autofluorescence profiles of both the oocyte and cumulus cells. In oocytes, cumulin significantly upregulated proteins involved in nuclear function. In cumulus cells, both OSFs elicited marked upregulation of a variety of metabolic processes (mostly anabolic), including lipid, nucleotide, and carbohydrate metabolism, whereas mitochondrial metabolic processes were downregulated. The mitochondrial changes were validated by functional assays confirming altered mitochondrial morphology, respiration, and content while maintaining ATP homeostasis. Collectively, these data demonstrate that cumulin and BMP15 remodel cumulus cell metabolism, instructing them to upregulate their anabolic metabolic processes, while routine cellular functions are minimized in the oocyte during maturation, in preparation for ensuing embryonic development.NEW & NOTEWORTHY Oocyte-secreted factors (OSFs) promote oocyte and cumulus cell cooperativity by altering the molecular composition of both cell types. OSFs downregulate protein catabolic processes and upregulate processes associated with DNA binding, translation, and ribosome assembly in oocytes. In cumulus cells, OSFs alter mitochondrial number, morphology, and function, and enhance metabolic plasticity by upregulating anabolic pathways. Hence, the oocyte via OSFs, instructs cumulus cells to increase metabolic processes on its behalf, thereby subduing oocyte metabolism.

Characterization of the molecular mechanisms that govern anti-Müllerian hormone synthesis and activity.

The roles of anti-Müllerian hormone (AMH) continue to expand, from its discovery as a critical factor in sex determination, through its identification as a regulator of ovarian folliculogenesis, its use in fertility clinics as a measure of ovarian reserve, and its emerging role in hypothalamic-pituitary function. In light of these actions, AMH is considered an attractive therapeutic target to address diverse reproductive needs, including fertility preservation. Here, we set out to characterize the molecular mechanisms that govern AMH synthesis and activity. First, we enhanced the processing of the AMH precursor to >90% by introducing more efficient proprotein convertase cleavage sites (RKKR or ISSRKKRSVSS [SCUT]). Importantly, enhanced processing corresponded with a dramatic increase in secreted AMH activity. Next, based on species differences across the AMH type II receptor-binding interface, we generated a series of human AMH variants and assessed bioactivity. AMHSCUT potency (EC50 4 ng/mL) was increased 5- or 10-fold by incorporating Gln484 Met/Leu535 Thr (EC50 0.8 ng/mL) or Gln484 Met/Gly533 Ser (EC50 0.4 ng/mL) mutations, respectively. Furthermore, the Gln484 Met/Leu535 Thr double mutant displayed enhanced efficacy, relative to AMHSCUT . Finally, we identified residues within the wrist pre-helix of AMH (Trp494 , Gln496 , Ser497 , and Asp498 ) that likely mediate type I receptor binding. Mutagenesis of these residues generated gain- (Trp494 Phe or Gln496 Leu) or loss- (Ser497 Ala) of function AMH variants. Surprisingly, combining activating type I and type II receptor mutations only led to modest additive increases in AMH potency/efficacy. Our study is the first to characterize AMH residues involved in type I receptor binding and suggests a step-wise receptor-complex assembly mechanism, in which enhancement in the affinity of the ligand for either receptor can increase AMH activity beyond the natural level.

Effects of lactate, super-GDF9, and low oxygen tension during bi-phasic in vitro maturation on the bioenergetic profiles of mouse cumulus-oocyte complex†.

In vitro maturation (IVM) is an alternative assisted reproductive technology with reduced hormone-related side effects and treatment burden compared to conventional IVF. Capacitation (CAPA)-IVM is a bi-phasic IVM system with improved clinical outcomes compared to standard monophasic IVM. Yet, CAPA-IVM efficiency compared to conventional IVF is still suboptimal in terms of producing utilizable blastocysts. Previously, we have shown that CAPA-IVM leads to a precocious increase in cumulus cell (CC) glycolytic activity during cytoplasmic maturation. In the current study, considering the fundamental importance of CCs for oocyte maturation and cumulus-oocyte complex (COC) microenvironment, we further analyzed the bioenergetic profiles of maturing CAPA-IVM COCs. Through a multi-step approach, we (i) explored mitochondrial function of the in vivo and CAPA-IVM matured COCs through real-time metabolic analysis with Seahorse analyzer, and to improve COC metabolism (ii) supplemented the culture media with lactate and/or super-GDF9 (an engineered form of growth differentiation factor 9) and (iii) reduced culture oxygen tension. Our results indicated that the pre-IVM step is delicate and prone to culture-related disruptions. Lactate and/or super-GDF9 supplementations failed to eliminate pre-IVM-induced stress on COC glucose metabolism and mitochondrial respiration. However, when performing pre-IVM culture under 5% oxygen tension, CAPA-IVM COCs showed similar bioenergetic profiles compared to in vivo matured counterparts. This is the first study providing real-time metabolic analysis of the COCs from a bi-phasic IVM system. The currently used analytical approach provides the quantitative measures and the rational basis to further improve IVM culture requirements.

Targeting activins and inhibins to treat reproductive disorders and cancer cachexia.

Although originally characterised as proteins involved in the control of reproductive function, activins, and to a lesser degree inhibins, are also important regulators of homeostasis in extragonadal tissues. Accordingly, disrupted inhibin/activin expression can have detrimental effects not only on fertility and fecundity but also on the regulation of muscle, fat and bone mass. Indeed, only recently, two complementary mouse models of inhibin designed to lack bioactivity/responsiveness revealed that inhibin A/B deficiency during pregnancy restricts embryo and fetal survival. Conversely, hyper-elevated levels of activin A/B, as are frequently observed in patients with advanced cancers, can not only promote gonadal tumour growth but also cancer cachexia. As such, it is not surprising that inhibin/activin genetic variations or altered circulating levels have been linked to reproductive disorders and cancer. Whilst some of the detrimental health effects associated with disrupted inhibin/activin levels can be attributed to accompanied changes in circulating follicle-stimulating hormone (FSH) levels, there is now abundant evidence that activins, in particular, have fundamental FSH-independent tissue homeostatic roles. Increased understanding of inhibin/activin activity, garnered over several decades, has enabled the development of targeted therapies with applications for both reproductive and extra-gonadal tissues. Inhibin- or activin-targeted technologies have been shown not just to enhance fertility and fecundity but also to reduce disease severity in models of cancer cachexia. Excitingly, these technologies are likely to benefit human medicine and be highly valuable to animal breeding and veterinary programmes.

Activin A level is associated with physical function in critically ill patients.

BACKGROUND: Activin A is a potent negative regulator of muscle mass elevated in critical illness. It is unclear whether muscle strength and physical function in critically ill humans are associated with elevated activin A levels. OBJECTIVES: The objective of this study was to investigate the relationship between serum activin A levels, muscle strength, and physical function at discharge from the intensive care unit (ICU) and hospital. METHODS: Thirty-six participants were recruited from two tertiary ICUs in Melbourne, Australia. Participants were included if they were mechanically ventilated for >48 h and expected to have a total ICU stay of >5 days. The primary outcome measure was the Six-Minute Walk Test distance at hospital discharge. Secondary outcome measures included handgrip strength, Medical Research Council Sum Score, Physical Function ICU Test Scored, Six-Minute Walk Test, and Timed Up and Go Test assessed throughout the hospital admission. Total serum activin A levels were measured daily in the ICU. RESULTS: High peak activin A was associated with worse Six-Minute Walk Test distance at hospital discharge (linear regression coefficient, 95% confidence interval, p-value: -91.3, -154.2 to -28.4, p = 0.007, respectively). Peak activin A concentration was not associated with the secondary outcome measures. CONCLUSIONS: Higher peak activin A may be associated with the functional decline of critically ill patients. Further research is indicated to examine its potential as a therapeutic target and a prospective predictor for muscle wasting in critical illness. STUDY REGISTRATION: ACTRN12615000047594.

'There's something about admitting that you are lonely' - prevalence, impact and solutions to loneliness in terminal illness: An explanatory sequential multi-methods study.

BACKGROUND: Loneliness is a prevalent societal issue and can impact on a person's physical and mental health. It is unclear how loneliness impacts on end of life experiences or how such feelings can be alleviated. AIM: To explore the perceived prevalence, impact and possible solutions to loneliness among people who are terminally ill and their carers in Northern Ireland through the lens of health and social care professionals. DESIGN: An explanatory multi-method study. SETTING/PARTICIPANTS: An online survey (n = 68, response rate 30%) followed by three online focus groups with palliative and end of life care health and social care professionals (n = 14). Data were analysed using descriptive statistics and thematic analysis. RESULTS: Loneliness was perceived by professionals as highly prevalent for people with a terminal illness (92.6%) and their carers (86.8%). Loneliness was considered a taboo subject and impacts on symptoms including pain and breathlessness and overall wellbeing at end of life. Social support was viewed as central towards alleviating feelings of loneliness and promoting connectedness at end of life. Four themes were identified: (1) the stigma of loneliness, (2) COVID-19: The loneliness pandemic (3) impact of loneliness across physical and mental health domains and (4) the power of social networks. CONCLUSION: There is a need for greater investment for social support initiatives to tackle experiences of loneliness at end of life. These services must be co-produced with people impacted by terminal illness to ensure they meet the needs of this population.

Inhibin Inactivation in Female Mice Leads to Elevated FSH Levels, Ovarian Overstimulation, and Pregnancy Loss.

Inhibins are members of the transforming growth factor-ß family, composed of a common α-subunit disulfide-linked to 1 of 2 ß-subunits (ßA in inhibin A or ßB in inhibin B). Gonadal-derived inhibin A and B act in an endocrine manner to suppress the synthesis of follicle-stimulating hormone (FSH) by pituitary gonadotrope cells. Roles for inhibins beyond the pituitary, however, have proven difficult to delineate because deletion of the inhibin α-subunit gene (Inha) results in unconstrained expression of activin A and activin B (homodimers of inhibin ß-subunits), which contribute to gonadal tumorigenesis and lethal cachectic wasting. Here, we generated mice with a single point mutation (Arg233Ala) in Inha that prevents proteolytic processing and the formation of bioactive inhibin. In vitro, this mutation blocked inhibin maturation and bioactivity, without perturbing activin production. Serum FSH levels were elevated 2- to 3-fold in InhaR233A/R233A mice due to the loss of negative feedback from inhibins, but no pathological increase in circulating activins was observed. While inactivation of inhibin A and B had no discernible effect on male reproduction, female InhaR233A/R233A mice had increased FSH-dependent follicle development and enhanced natural ovulation rates. Nevertheless, inhibin inactivation resulted in significant embryo-fetal resorptions and severe subfertility and was associated with disrupted maternal ovarian function. Intriguingly, heterozygous Inha+/R233A females had significantly enhanced fecundity, relative to wild-type littermates. These studies have revealed novel effects of inhibins in the establishment and maintenance of pregnancy and demonstrated that partial inactivation of inhibin A/B is an attractive approach for enhancing female fertility.

Human INHBB Gene Variant (c.1079T>C:p.Met360Thr) Alters Testis Germ Cell Content, but Does Not Impact Fertility in Mice.

Testicular-derived inhibin B (α/ß B dimers) acts in an endocrine manner to suppress pituitary production of follicle-stimulating hormone (FSH), by blocking the actions of activins (ß A/B/ß A/B dimers). Previously, we identified a homozygous genetic variant (c.1079T>C:p.Met360Thr) arising from uniparental disomy of chromosome 2 in the INHBB gene (ß B-subunit of inhibin B and activin B) in a man suffering from infertility (azoospermia). In this study, we aimed to test the causality of the p.Met360Thr variant in INHBB and testis function. Here, we used CRISPR/Cas9 technology to generate InhbbM364T/M364T mice, where mouse INHBB p.Met364 corresponds with human p.Met360. Surprisingly, we found that the testes of male InhbbM364T/M364T mutant mice were significantly larger compared with those of aged-matched wildtype littermates at 12 and 24 weeks of age. This was attributed to a significant increase in Sertoli cell and round spermatid number and, consequently, seminiferous tubule area in InhbbM364T/M364T males compared to wildtype males. Despite this testis phenotype, male InhbbM364T/M364T mutant mice retained normal fertility. Serum hormone analyses, however, indicated that the InhbbM364T variant resulted in reduced circulating levels of activin B but did not affect FSH production. We also examined the effect of this p.Met360Thr and an additional INHBB variant (c.314C>T: p.Thr105Met) found in another infertile man on inhibin B and activin B in vitro biosynthesis. We found that both INHBB variants resulted in a significant disruption to activin B in vitro biosynthesis. Together, this analysis supports that INHBB variants that limit activin B production have consequences for testis composition in males.

Effect of cumulin and super-GDF9 in standard and biphasic mouse IVM.

PURPOSE: In vitro maturation (IVM) is a technology that generates mature oocytes following culture of immature cumulus-oocyte complexes (COC) in vitro. IVM is characterized by minimal patient stimulation, making it attractive for certain patient groups. Recently, a biphasic IVM system, capacitation (CAPA)-IVM, has shown improved clinical outcomes relative to standard IVM; however, it remains less efficient than IVF. This study assessed whether supplementation of CAPA-IVM culture media with the novel TGFß superfamily proteins cumulin and super-GDF9 improves subsequent mouse embryo development. METHODS: Immature mouse COCs were cultured by standard IVM or biphasic IVM ± cumulin or super-GDF9. RESULTS: Both cumulin and super-GDF9 in standard IVM significantly improved day-6 blastocyst rate (53.9% control, 73.6% cumulin, 70.4% super-GDF9; p = 0.006; n = 382-406 oocytes). Cumulin or super-GDF9 in CAPA-IVM did not alter embryo yield or blastocyst cell allocation in an unstimulated model. Moreover, cumulin did not alter these outcomes in a mild PMSG stimulation model. Cumulin in CAPA-IVM significantly increased cumulus cell expression of cumulus expansion genes (Ptgs2, Ptx3, Adamts1, Gfat2) and decreased Lhr expression relative to control. However, cumulin-induced mRNA expression of cumulus cell (Ptgs2, Ptx3) and oocyte genes (Gdf9, Bmp15, Oct4, Stella) in CAPA-IVM remained significantly lower than that of in vivo matured cells. CONCLUSION: Cumulin did not provide an additional beneficial effect in biphasic IVM in terms of blastocyst yield and cell allocation; however in standard IVM, cumulin and super-GDF9 significantly improve oocyte developmental competence.

Relationship Between Strength, Athletic Performance, and Movement Skill in Adolescent Girls.

ABSTRACT: Sommerfield, LM, Harrison, CB, Whatman, CS, and Maulder, PS. Relationship between strength, athletic performance, and movement skill in adolescent girls. J Strength Cond Res 36(3): 674-679, 2022-Muscular strength in youth has been linked to health and physical benefits, enhanced movement skill, and an active lifestyle in adulthood. However, the relationship between maximum strength, athletic performance, and movement skill in youth females remains unclear. The purpose was to examine the relationship between maximum strength, athletic performance, and movement skill and determine whether differences exist between strong girls (SGs), average girls (AGs), and weak girls (WGs). One hundred four girls (age 14.0 ± 0.6 years, height 162.6 ± 5.9 cm, body mass 57.3 ± 9.7 cm) from a girls' secondary school performed an isometric midthigh pull (IMTP), double- and single-leg (right leg = R, left leg = L) countermovement jump, 10- and 20-m sprints, a drop vertical jump ,and the back squat assessment. Significance was set at p < 0.01 for correlations and p < 0.05 for one-way analysis of variance. Correlations revealed IMTP had significant small to large relationships with all performance variables (r = 0.27-0.62) except right-leg countermovement jump and left-leg countermovement jump height (r = 0.17-0.23). Relative IMTP had significant moderate to large relationships with all performance variables (r = 0.32-0.60). There were significant differences between strength groups for all performance measures. Strong girls had significantly faster sprint times than AGs. In addition, SGs and AGs performed significantly better than WGs in all assessments. The results of this study demonstrate the importance of strength for athletic performance and movement skill in adolescent girls.

TGFBR3L is an inhibin B co-receptor that regulates female fertility.

Follicle-stimulating hormone (FSH), a key regulator of ovarian function, is often used in infertility treatment. Gonadal inhibins suppress FSH synthesis by pituitary gonadotrope cells. The TGFß type III receptor, betaglycan, is required for inhibin A suppression of FSH. The inhibin B co-receptor was previously unknown. Here, we report that the gonadotrope-restricted transmembrane protein, TGFBR3L, is the elusive inhibin B co-receptor. TGFBR3L binds inhibin B but not other TGFß family ligands. TGFBR3L knockdown or overexpression abrogates or confers inhibin B activity in cells. Female Tgfbr3l knockout mice exhibit increased FSH levels, ovarian follicle development, and litter sizes. In contrast, female mice lacking both TGFBR3L and betaglycan are infertile. TGFBR3L's function and cell-specific expression make it an attractive new target for the regulation of FSH and fertility.

The effects of strength vs. plyometric training on sprinting kinetics in post peak height velocity (PHV) female athletes.

Speed is a crucial factor for overall athletic development. While researchers have shown strength and plyometric training to improve sprinting speed in some adult and youth populations, no studies have compared the effects of strength and plyometric training on sprinting speed in young females. Fifty-two young females were divided into three groups and trained for 7 weeks, twice a week; strength training (n = 16, age 13.36 ± 0.84), plyometric training (n = 21, age 13.38 ± 0.75) and a physical education class as a control group (n = 15, age 13.95 ± 0.54). Participants were tested for sprinting performance and horizontal force (Fo), maximum velocity (Vmax) and maximum horizontal power (Pmax) metrics over 30 m distance, isometric strength and unilateral horizontal jump distance before and after the intervention. Both the strength and plyometric groups significantly improved all performance variables (p < 0.05). The strength group significantly improved 10 m split time (6.76%; Hedge's g = 0.65) and Fo (18.98%; g = 0.67), whereas the plyometric group significantly improved Vmax (4.91%; g = 0.50) and Pmax (7.91%; g = 0.31). The findings of this study suggest that both strength and plyometric training can improve sprinting kinetics, jumping performance and overall strength in young females.

Resisted Sprint Training in Youth: The Effectiveness of Backward vs. Forward Sled Towing on Speed, Jumping, and Leg Compliance Measures in High-School Athletes.

ABSTRACT: Uthoff, A, Oliver, J, Cronin, J, Winwood, P, Harrison, C, and Lee, JE. Resisted sprint training in youth: the effectiveness of backward vs. forward sled towing on speed, jumping, and leg compliance measures in high-school athletes. J Strength Cond Res 35(8): 2205-2212, 2021-Resisted sprinting (RS) is a popular training method used to enhance sprinting performance in youth. However, research has only explored the effects of forward RS (FRS) training. We examined the effects of FRS and backward RS (BRS) and compared these with a traditional physical education curriculum (CON). One hundred fifteen boys (age 13-15 years) were matched for maturity and allocated to either an FRS (n = 34), BRS (n = 46), or CON (n = 35) group. Training groups towed progressively overloaded sleds (20-55% body mass) 2 d·wk-1 for 8 weeks. Pre-training and post-training data were collected for sprinting times over 10 and 20 m, countermovement jump (CMJ) height, and leg stiffness (KN). Performance remained unchanged for the CON group (all p > 0.05), whereas all variables significantly improved (p < 0.05) after BRS, and all but 10-m performance improved after FRS. Compared with the CON, BRS and FRS significantly (p > 0.05) improved CMJ (Effect size [ES] = 0.67 and 0.38) and KN (ES = 0.94 and 0.69), respectively. No differences were found between training groups. The probabilities of improving sprinting performance after BRS (∼70%) were on average ∼10 and ∼8% better than the FRS and CON groups, respectively. The BRS and FRS showed similar probabilities of improving CMJ (75 and 79%) and KN (80 and 81%), respectively, over the CON group. It seems that BRS may be a means to improve sprint performance, and regardless of direction, RS seems to be a beneficial method for improving jumping height and leg stiffness in youth male athletes.

Structure of AMH bound to AMHR2 provides insight into a unique signaling pair in the TGF-ß family.

Anti-Müllerian hormone (AMH), or Müllerian-inhibiting substance, is a protein hormone that promotes Müllerian duct regression during male fetal sexual differentiation and regulation of folliculogenesis in women. AMH is a member of the transforming growth factor beta (TGF-ß) family, which has evolved to signal through its own dedicated type II receptor, AMH receptor type II (AMHR2). Structures of other TGF-ß family members have revealed how ligands infer specificity for their cognate receptors; however, it is unknown how AMH binds AMHR2 at the molecular level. Therefore, in this study, we solved the X-ray crystal structure of AMH bound to the extracellular domain of AMHR2 to a resolution of 2.6Å. The structure reveals that while AMH binds AMHR2 in a similar location to Activin and BMP ligand binding to their type II receptors, differences in both AMH and AMHR2 account for a highly specific interaction. Furthermore, using an AMH responsive cell-based luciferase assay, we show that a conformation in finger 1 of AMHR2 and a salt bridge formed by K534 on AMH and D81/E84 of AMHR2 are key to the AMH/AMHR2 interaction. Overall, our study highlights how AMH engages AMHR2 using a modified paradigm of receptor binding facilitated by modifications to the three-finger toxin fold of AMHR2. Furthermore, understanding these elements contributing to the specificity of binding will help in the design of agonists or antagonists or the selection of antibody therapies.

Effects of Combined Resistance Training and Weightlifting on Injury Risk Factors and Resistance Training Skill of Adolescent Males.

ABSTRACT: Pichardo, AW, Oliver, JL, Harrison, CB, Maulder, PS, Lloyd, RS, and Kandoi, R. Effects of combined resistance training and weightlifting on injury risk factors and resistance training skill of adolescent males. J Strength Cond Res 35(12): 3370-3377, 2021-The purpose of this study was to investigate the effects of resistance training with or without weightlifting on risk factors for injury and resistance training skill in circa-peak height velocity boys. Sixty-seven boys (aged 12-14 years) from a local secondary school were divided into 3 groups: combined resistance training (CRT), combined resistance training with weightlifting movements (CRT&WL), or a control group (CON). Experimental groups completed twice-weekly training programs over the course of an academic year. The tuck jump assessment, asymmetry measures for single-leg horizontal jump, isometric midthigh pull, and the Star Excursion Balance Test, and resistance training skill were measured pre-, mid-, and post-intervention. Only the CRT group significantly improved tuck jump assessment score pre- to post-test (p = 0.006, -20.4%, d = -0.39) but there were no clear effects on asymmetry measures for any group. Both groups significantly improved resistance training skill from pre- to post-test (CRT&WL: p = 0.002, 17.6%, d = 1.00; CRT: p = 0.026, 9.2%, d = 0.53). This study suggests that a school-based CRT program may provide significant improvements in jump landing kinematics, whereas the inclusion of weightlifting movements may provide greater improvements in resistance training skill.

Fibroblast Nox2 (NADPH Oxidase-2) Regulates ANG II (Angiotensin II)-Induced Vascular Remodeling and Hypertension via Paracrine Signaling to Vascular Smooth Muscle Cells.

OBJECTIVE: The superoxide-generating Nox2 (NADPH oxidase-2) is expressed in multiple cell types. Previous studies demonstrated distinct roles for cardiomyocyte, endothelial cell, and leukocyte cell Nox2 in ANG II (angiotensin II)-induced cardiovascular remodeling. However, the in vivo role of fibroblast Nox2 remains unclear. Approach and Results: We developed a novel mouse model with inducible fibroblast-specific deficiency of Nox2 (fibroblast-specific Nox2 knockout or Fibro-Nox2KO mice) and investigated the responses to chronic ANG II stimulation. Fibro-Nox2KO mice showed no differences in basal blood pressure or vessel wall morphology, but the hypertensive response to ANG II infusion (1.1 mg/[kg·day] for 14 days) was substantially reduced as compared to control Nox2-Flox littermates. This was accompanied by a significant attenuation of aortic and resistance vessel remodeling. The conditioned medium of ANG II-stimulated primary fibroblasts induced a significant increase in vascular smooth muscle cell growth, which was inhibited by the short hairpin RNA (shRNA)-mediated knockdown of fibroblast Nox2. Mass spectrometric analysis of the secretome of ANG II-treated primary fibroblasts identified GDF6 (growth differentiation factor 6) as a potential growth factor that may be involved in these effects. Recombinant GDF6 induced a concentration-dependent increase in vascular smooth muscle cell growth while chronic ANG II infusion in vivo significantly increased aortic GDF6 protein levels in control mice but not Fibro-Nox2KO animals. Finally, silencing GDF6 in fibroblasts prevented the induction of vascular smooth muscle cell growth by fibroblast-conditioned media in vitro. CONCLUSIONS: These results indicate that fibroblast Nox2 plays a crucial role in the development of ANG II-induced vascular remodeling and hypertension in vivo. Mechanistically, fibroblast Nox2 may regulate paracrine signaling to medial vascular smooth muscle cells via factors, such as GDF6.

Potential treatment of keloid pathogenesis with follistatin 288 by blocking the activin molecular pathway.

Keloids are benign tumours caused by abnormal wound healing driven by increased expression of cytokines, including activin A. This study compared effects of activins on normal and keloid-derived human dermal fibroblasts and investigated a novel treatment for keloids using follistatin. Normal skin and keloid tissue samples from 11 patients were used to develop primary fibroblast cultures, which were compared in terms of their histology and relevant gene (qRT-PCR and RNAseq) and protein (ELISA) expression. Activin A (INHBA) and connective tissue growth factor (CTGF) gene expression were significantly upregulated in keloid fibroblasts, as was activin A protein expression in cell lysates and culture medium. Activator protein 1 inhibitor (SR11302) significantly decreased INHBA and CTGF expression in keloid fibroblasts and a single treatment of follistatin over 5 days significantly inhibited activin and various matrix-related genes in keloid fibroblasts when compared to controls. Follistatin, by binding activin A, suppressed CTGF expression suggesting a novel therapeutic role in managing keloids and perhaps other fibrotic diseases.

TMEPAI/PMEPA1 Is a Positive Regulator of Skeletal Muscle Mass.

Inhibition of myostatin- and activin-mediated SMAD2/3 signaling using ligand traps, such as soluble receptors, ligand-targeting propeptides and antibodies, or follistatin can increase skeletal muscle mass in healthy mice and ameliorate wasting in models of cancer cachexia and muscular dystrophy. However, clinical translation of these extracellular approaches targeting myostatin and activin has been hindered by the challenges of achieving efficacy without potential effects in other tissues. Toward the goal of developing tissue-specific myostatin/activin interventions, we explored the ability of transmembrane prostate androgen-induced (TMEPAI), an inhibitor of transforming growth factor-ß (TGF-ß1)-mediated SMAD2/3 signaling, to promote growth, and counter atrophy, in skeletal muscle. In this study, we show that TMEPAI can block activin A, activin B, myostatin and GDF-11 activity in vitro. To determine the physiological significance of TMEPAI, we employed Adeno-associated viral vector (AAV) delivery of a TMEPAI expression cassette to the muscles of healthy mice, which increased mass by as much as 30%, due to hypertrophy of muscle fibers. To demonstrate that TMEPAI mediates its effects via inhibition of the SMAD2/3 pathway, tibialis anterior (TA) muscles of mice were co-injected with AAV vectors expressing activin A and TMEPAI. In this setting, TMEPAI blocked skeletal muscle wasting driven by activin-induced phosphorylation of SMAD3. In a model of cancer cachexia associated with elevated circulating activin A, delivery of AAV:TMEPAI into TA muscles of mice bearing C26 colon tumors ameliorated the muscle atrophy normally associated with cancer progression. Collectively, the findings indicate that muscle-directed TMEPAI gene delivery can inactivate the activin/myostatin-SMAD3 pathway to positively regulate muscle mass in healthy settings and models of disease.

Engineering the Ovarian Hormones Inhibin A and Inhibin B to Enhance Synthesis and Activity.

Ovarian-derived inhibin A and inhibin B (heterodimers of common α- and differing ß-subunits) are secreted throughout the menstrual cycle in a discordant pattern, with smaller follicles producing inhibin B, whereas the dominant follicle and corpus luteum produce inhibin A. The classical function for endocrine inhibins is to block signalling by activins (homodimers of ß-subunits) in gonadotrope cells of the anterior pituitary and, thereby, inhibit the synthesis of FSH. Whether inhibin A and inhibin B have additional physiological functions is unknown, primarily because producing sufficient quantities of purified inhibins, in the absence of contaminating activins, for preclinical studies has proven extremely difficult. Here, we describe novel methodology to enhance inhibin A and inhibin B activity and to produce these ligands free of contaminating activins. Using computational modeling and targeted mutagenesis, we identified a point mutation in the activin ß A-subunit, A347H, which completely disrupted activin dimerization and activity. Importantly, this ß A-subunit mutation had minimal effect on inhibin A bioactivity. Mutation of the corresponding residue in the inhibin ß B-subunit, G329E, similarly disrupted activin B synthesis/activity without affecting inhibin B production. Subsequently, we enhanced inhibin A potency by modifying the binding site for its co-receptor, betaglycan. Introducing a point mutation into the α-subunit (S344I) increased inhibin A potency ~12-fold. This study has identified a means to eliminate activin A/B interference during inhibin A/B production, and has facilitated the generation of potent inhibin A and inhibin B agonists for physiological exploration.