Prevalence, patient characteristics and treatment patterns among SLE-PAH patients in real-world clinical practice: A retrospective analysis of Medical Data Vision Database in Japan.

OBJECTIVE: Real-world evidence regarding prevalence, patient characteristics and treatment patterns for pulmonary arterial hypertension (PAH) related to systemic lupus erythematosus (SLE) in Japan is limited. METHODS: We conducted a retrospective study analysing Japan's Medical Data Vision (MDV) database from April-2008 to September-2020. Prevalence, incidence, patient characteristics, treatment patterns and use of vasodilators by treatment line were evaluated. RESULTS: Prevalence of PAH was 0.392% in SLE patients (n=114/29,077). Cumulative incidence was 0.53% (3-years (y)) and 0.77% (5y). Of 114 SLE-PAH patients, 49% developed PAH <1 year from SLE diagnosis. SLE-PAH patients were more female (88% vs. 72%), had lower mean age at SLE diagnosis (53y vs. 56y) and more severe SLE (61% vs 25%), versus non-PAH SLE patients. Glucocorticoids (58%) and vasodilators (27%) were preferred first-line monotherapy for SLE-PAH. Glucocorticoids+immunosuppressants (19%) was predominant first-line combination therapy. Endothelin receptor antagonists (40% and 44%) and nitric oxide analogues (31% and 40%) were dominant first- and second-line vasodilators. CONCLUSION: SLE-PAH patients were more females, younger at diagnosis, had more severe SLE than non-PAH SLE patients. Most were diagnosed <1 year of SLE diagnosis. In Japan's real-world practice, initial treatment goal is SLE management, while vasodilators are preferred in advanced disease, as per MDV database.

Suppression of Sertoli cell tumour development during the first wave of spermatogenesis in inhibin α-deficient mice.

A dynamic partnership between follicle-stimulating hormone (FSH) and activin is required for normal Sertoli cell development and fertility. Disruptions to this partnership trigger Sertoli cells to deviate from their normal developmental pathway, as observed in inhibin α-knockout (Inha-KO) mice, which feature Sertoli cell tumours in adulthood. Here, we identified the developmental windows by which adult Sertoli cell tumourigenesis is most FSH sensitive. FSH was suppressed for 7 days in Inha-KO mice and wild-type littermates during the 1st, 2nd or 4th week after birth and culled in the 5th week to assess the effect on adult Sertoli cell development. Tumour growth was profoundly reduced in adult Inha-KO mice in response to FSH suppression during Weeks 1 and 2, but not Week 4. Proliferative Sertoli cells were markedly reduced in adult Inha-KO mice following FSH suppression during Weeks 1, 2 or 4, resulting in levels similar to those in wild-type mice, with greatest effect observed at the 2 week time point. Apoptotic Sertoli cells increased in adult Inha-KO mice after FSH suppression during Week 4. In conclusion, acute FSH suppression during the 1st or 2nd week after birth in Inha-KO mice profoundly suppresses Sertoli cell tumour progression, probably by inhibiting proliferation in the adult, with early postnatal Sertoli cells being most sensitive to FSH action.

Elevated expression of activins promotes muscle wasting and cachexia.

In models of cancer cachexia, inhibiting type IIB activin receptors (ActRIIBs) reverse muscle wasting and prolongs survival, even with continued tumor growth. ActRIIB mediates signaling of numerous TGF-ß proteins; of these, we demonstrate that activins are the most potent negative regulators of muscle mass. To determine whether activin signaling in the absence of tumor-derived factors induces cachexia, we used recombinant serotype 6 adeno-associated virus (rAAV6) vectors to increase circulating activin A levels in C57BL/6 mice. While mice injected with control vector gained ~10% of their starting body mass (3.8±0.4 g) over 10 wk, mice injected with increasing doses of rAAV6:activin A exhibited weight loss in a dose-dependent manner, to a maximum of -12.4% (-4.2±1.1 g). These reductions in body mass in rAAV6:activin-injected mice correlated inversely with elevated serum activin A levels (7- to 24-fold). Mechanistically, we show that activin A reduces muscle mass and function by stimulating the ActRIIB pathway, leading to deleterious consequences, including increased transcription of atrophy-related ubiquitin ligases, decreased Akt/mTOR-mediated protein synthesis, and a profibrotic response. Critically, we demonstrate that the muscle wasting and fibrosis that ensues in response to excessive activin levels is fully reversible. These findings highlight the therapeutic potential of targeting activins in cachexia.

Hypoxia-mediated carbohydrate metabolism and transport promote early-stage murine follicle growth and survival.

Oxygen tension is critical for follicle growth and metabolism, especially for early-stage follicles, where vascularity is limited. Its role and underlying mechanism in the in vitro activation and maturation of immature to ovulatory follicles is largely unknown. In this study, early secondary (110 µm) murine follicles were isolated and encapsulated in alginate hydrogels to replicate the in vivo environment of the growing/maturing follicle. Encapsulated follicles were cultured for 8 days at either 2.5 or 20% O2. Survival (2.6-fold) and growth (1.2-fold) were significantly higher for follicles cultured at 2.5% compared with 20% O2. Using a mouse hypoxia-signaling pathway qRT-PCR array and GeneGo Metacore analysis, we found that direct target genes of the hypoxia-activated HIF1-complex were significantly upregulated in follicles cultured for 8 days at 2.5% compared with 20% O2, including the carbohydrate transport and metabolism genes Slc2a3, Vegfa, Slc2a1, Edn1, Pgk1, Ldha, and Hmox1. Other upregulated genes included carbohydrate transporters (Slc2a1, Slc2a3, and Slc16a3) and enzymes essential for glycolysis (Pgk1, Hmox1, Hk2, Gpi1, Pfkl, Pfkp, Aldoa, Gapdh, Pgam1, Eno1, Pkm2, and Ldha). For follicles cultured at 2.5% O2, a 7.2-fold upregulation of Vegfa correlated to an 18-fold increase in VEGFA levels, and a 3.2-fold upregulation of Ldha correlated to a 4.8-fold increase in lactate levels. Both VEGFA and lactate levels were significantly higher in follicles cultured at 2.5% compared with 20% O2. Therefore, enhanced hypoxia-mediated glycolysis is essential for growth and survival of early secondary follicles and provides vital insights into improving in vitro culture conditions.

Promoting extracellular matrix remodeling via ascorbic acid enhances the survival of primary ovarian follicles encapsulated in alginate hydrogels.

The in vitro growth of ovarian follicles is an emerging technology for fertility preservation. Various strategies support the culture of secondary and multilayer follicles from various species including mice, non-human primate, and human; however, the culture of early stage (primary and primordial) follicles, which are more abundant in the ovary and survive cryopreservation, has been limited. Hydrogel-encapsulating follicle culture systems that employed feeder cells, such as mouse embryonic fibroblasts (MEFs), stimulated the growth of primary follicles (70-80 µm); yet, survival was low and smaller follicles (<70 µm) rapidly lost structure and degenerated. These morphologic changes were associated with a breakdown of the follicular basement membrane; hence, this study investigated ascorbic acid based on its role in extracellular matrix (ECM) deposition/remodeling for other applications. The selection of ascorbic acid was further supported by a microarray analysis that suggested a decrease in mRNA levels of enzymes within the ascorbate pathway between primordial, primary, and secondary follicles. The supplementation of ascorbic acid (50 µg/mL) significantly enhanced the survival of primary follicles (<80 µm) cultured in alginate hydrogels, which coincided with improved structural integrity. Follicles developed antral cavities and increased to diameters exceeding 250 µm. Consistent with improved structural integrity, the gene/protein expression of ECM and cell adhesion molecules was significantly changed. This research supports the notion that modifying the culture environment (medium components) can substantially enhance the survival and growth of early stage follicles.

Activin-ß(c) reduces reproductive tumour progression and abolishes cancer-associated cachexia in inhibin-deficient mice.

Activins are involved in the regulation of a diverse range of physiological processes including development, reproduction, and fertility, and have been implicated in the progression of cancers. Bioactivity is regulated by the inhibin α-subunit and by an activin-binding protein, follistatin. The activin-ß(C) subunit was not considered functionally significant in this regard due to an absence of phenotype in knockout mice. However, activin-ß(C) forms heterodimers with activin-ß(A) and activin-C antagonizes activin-A in vitro. Thus, it is proposed that overexpression, rather than loss of activin-ß(C) , regulates activin-A bioactivity. In order to prove biological efficacy, inhibin α-subunit knockout mice (α-KO) were crossed with mice overexpressing activin-ß(C) (ActC++). Deletion of inhibin leads to Sertoli and granulosa cell tumours, increased activin-A, and cancer-associated cachexia. Therefore, cachexia and reproductive tumour development should be modulated in α-KO/ActC++ mice, where excessive activin-A is the underlying cause. Accordingly, a reduction in activin-A, no significant weight loss, and reduced incidence of reproductive tumours were evident in α-KO/ActC++ mice. Overexpression of activin-ß(C) antagonized the activin signalling cascade; thus, the tumourigenic effects of activin-A were abrogated. This study provides proof of the biological relevance of activin-ß(C) . Being a regulator of activin-A, it is able to abolish cachexia and modulate reproductive tumour development in α-KO mice.

Comparative adherence of macitentan versus ambrisentan and bosentan in Australian patients with pulmonary arterial hypertension: a retrospective real-world database study.

AIM: Bosentan, ambrisentan, and macitentan are endothelin receptor antagonists (ERAs), currently available in Australia for treatment of pulmonary arterial hypertension (PAH). This study assessed the comparative adherence of these ERAs for PAH in Australian patients. METHODS: This retrospective, observational study used data for adults with PAH from the Services Australia 10% Pharmaceuticals Benefits Scheme (PBS) dataset (01/2006-10/2020). The primary outcome was treatment adherence (i.e. receiving ≥80% of ERA doses over 12 months). Secondary outcomes were time to treatment change (add-on or switch) and overall survival. RESULTS: The study included 436 patients who took bosentan (n = 200), ambrisentan (n = 69), or macitentan (n = 167). Treatment adherence was significantly greater in patients who received macitentan (65.3%) versus ambrisentan (56.5%) and bosentan (58.0%), with odds ratios (ORs; 95% CI) of 0.51 (0.30-0.88; p = 0.016) for bosentan versus macitentan and 0.48 (0.24-0.96; p = 0.037) for ambrisentan versus macitentan. The median time to treatment change was 47.2 and 43.4 months for bosentan and ambrisentan, respectively (not calculated for macitentan because of insufficient duration of data). LIMITATIONS AND CONCLUSIONS: Real-world data for Australian patients with PAH showed that treatment adherence for ERAs was suboptimal. Adherence was higher for macitentan compared with ambrisentan and bosentan.

Inhibin α-subunit N terminus interacts with activin type IB receptor to disrupt activin signaling.

Inhibin is a heterodimeric peptide hormone produced in the ovary that antagonizes activin signaling and FSH synthesis in the pituitary. The inhibin ß-subunit interacts with the activin type II receptor (ActRII) to functionally antagonize activin. The inhibin α-subunit mature domain (N terminus) arose relatively early during the evolution of the hormone, and inhibin function is decreased by an antibody directed against the α-subunit N-terminal extension region or by deletion of the N-terminal region. We hypothesized that the α-subunit N-terminal extension region interacts with the activin type I receptor (ALK4) to antagonize activin signaling in the pituitary. Human or chicken free α-subunit inhibited activin signaling in a pituitary gonadotrope-derived cell line (LßT2) in a dose-dependent manner, whereas an N-terminal extension deletion mutant did not. An α-subunit N-terminal peptide, but not a control peptide, was able to inhibit activin A signaling and decrease activin-stimulated FSH synthesis. Biotinylated inhibin A, but not activin A, bound ALK4. Soluble ALK4-ECD bioneutralized human free α-subunit in LßT2 cells, but did not affect activin A function. Competitive binding ELISAs with N-terminal mutants and an N-terminal region peptide confirmed that this region is critical for direct interaction of the α-subunit with ALK4. These data expand our understanding of how endocrine inhibin achieves potent antagonism of local, constitutive activin action in the pituitary, through a combined mechanism of competitive binding of both ActRII and ALK4 by each subunit of the inhibin heterodimer, in conjunction with the co-receptor betaglycan, to block activin receptor-ligand binding, complex assembly, and downstream signaling.

Supplemented αMEM/F12-based medium enables the survival and growth of primary ovarian follicles encapsulated in alginate hydrogels.

Hydrogel-encapsulating culture systems for ovarian follicles support the in vitro growth of secondary follicles from various species including mouse, non-primate human, and human; however, the growth of early stage follicles (primary and primordial) has been limited. While encapsulation maintains the structure of early stage follicles, feeder cell populations, such as mouse embryonic fibroblasts (MEFs), are required to stimulate growth and development. Hence, in this report, we investigated feeder-free culture environments for early stage follicle development. Mouse ovarian follicles were encapsulated within alginate hydrogels and cultured in various growth medium formulations. Initial studies employed embryonic stem cell medium formulations as a tool to identify factors that influence the survival, growth, and meiotic competence of early stage follicles. The medium formulation that maximized survival and growth was identified as αMEM/F12 supplemented with fetuin, insulin, transferrin, selenium, and follicle stimulating hormone (FSH). This medium stimulated the growth of late primary (average initial diameter of 80 µm) and early secondary (average initial diameter of 90 µm) follicles, which developed antral cavities and increased to terminal diameters exceeding 300 µm in 14 days. Survival ranged from 18% for 80 µm follicles to 36% for 90 µm follicles. Furthermore, 80% of the oocytes from surviving follicles with an initial diameter of 90-100 µm underwent germinal vesicle breakdown (GVBD), and the percentage of metaphase II (MII) eggs was 50%. Follicle/oocyte growth and GVBD/MII rates were not significantly different from MEF co-culture. Survival was reduced relative to MEF co-culture, yet substantially increased relative to the control medium that had been previously used for secondary follicles. Continued development of culture medium could enable mechanistic studies of early stage folliculogenesis and emerging strategies for fertility preservation.

Risk Factors for Pulmonary Arterial Hypertension in Patients With Systemic Lupus Erythematosus: A Systematic Review and Expert Consensus.

OBJECTIVE: This study aimed to identify risk factors associated with the development of pulmonary arterial hypertension (PAH) in patients with systemic lupus erythematosus (SLE). METHODS: We conducted a systematic literature review of studies focusing on adult patients classified as having SLE-related PAH by searching the electronic databases Embase, Medline, Medline in-progress, Wanfang, China National Knowledge Infrastructure, Ichushi Web, Kmbase, and KoreaMed. Based on the findings, we conducted a Delphi survey to build expert consensus on issues related to screening for PAH in patients with SLE and on the importance and feasibility of measuring the identified factors in clinical practice. RESULTS: We included 21 eligible studies for data synthesis. Sixteen factors were associated with an increased risk of SLE-PAH: pericardial effusion, serositis, longer duration of SLE, arthritis, acute and subacute cutaneous lupus, scleroderma pattern on nailfold capillaroscopy, diffusion capacity of carbon monoxide in the lungs (DLCO) <70% predicted, interstitial lung disease, thrombocytopenia, and seven serological factors. Six factors were associated with a decreased risk of SLE-PAH: malar/acute rash, hematologic disorder, renal disorder, higher Systemic Lupus Erythematosus Disease Activity Index score, and two serological factors. Among these, there were six risk factors on which the panelists reached strong or general consensus (peak tricuspid regurgitation velocity on echocardiography >2.8 m/s, pericardial effusion, DLCO <70% predicted, scleroderma pattern on nailfold capillaroscopy, brain natriuretic peptide >50 ng/l, and N-terminal pro-brain natriuretic peptide >300 ng/l). The Delphi panel confirmed the need for a screening tool to identify patients with SLE at high risk of developing PAH and provided consensus on the importance and/or practicality of measuring the identified factors. CONCLUSION: The risk factors we identified could be used in a screening algorithm to identify patients with SLE with a high risk of developing PAH to facilitate early diagnosis, which could improve prognosis and management of these patients.

Comparative Treatment Persistence and Adherence to Endothelin Receptor Antagonists Among Patients with Pulmonary Arterial Hypertension in Japan: A Real-World Administrative Claims Database Study.

INTRODUCTION: Real-world data on the comparative effectiveness of endothelin receptor antagonists (ERAs; macitentan, bosentan, ambrisentan) for pulmonary arterial hypertension (PAH), particularly in Asian countries, are scarce. We evaluated the persistence of these ERAs before and after macitentan approval in Japan (2015). METHODS: We used real-world data from the Japanese Medical Data Vision administrative claims database between April 2008 and November 2020. Patients with PAH were identified from the dataset. Persistence to ERA treatment before and after approval of macitentan in Japan was defined as the time between start of the index ERA and treatment discontinuation or death. Propensity score adjustment was applied to minimize confounding effects among treatment groups. RESULTS: In the pre-macitentan approval cohort, 153 and 51 patients received bosentan and ambrisentan, respectively. In the post-macitentan approval cohort, 331, 284, and 91 patients received macitentan, bosentan, and ambrisentan, respectively. Unadjusted median persistence for ambrisentan- and bosentan-treated patients was 19 and 10 months, respectively (adjusted HR 0.87 [95% CI 0.61-1.24]; P = 0.434 [bosentan as reference]). In the post-macitentan approval cohort, unadjusted median persistence was 18 months for macitentan-treated patients versus 6 and 8 months for ambrisentan- and bosentan-treated patients, respectively. Adjusted HRs for ambrisentan and bosentan were 1.48 (95% CI 1.12-1.95; P = 0.006) and 1.63 (95% CI 1.30-2.04; P < 0.001 [macitentan as reference]), respectively. CONCLUSIONS: Real-world data for Japanese patients with PAH showed that persistence was significantly higher for macitentan, versus ambrisentan and bosentan, since its approval.

Two distinct regions of latency-associated peptide coordinate stability of the latent transforming growth factor-beta1 complex.

Transforming growth factor-beta1 (TGF-beta1) is secreted as part of an inactive complex consisting of the mature dimer, the TGF-beta1 propeptide (latency-associated peptide (LAP)), and latent TGF-beta-binding proteins. Using in vitro mutagenesis, we identified the regions of LAP that govern the cooperative assembly and stability of the latent TGF-beta1 complex. Initially, hydrophobic LAP residues (Ile(53), Leu(54), Leu(57), and Leu(59)), which form a contiguous epitope on one surface of an amphipathic alpha-helix, interact with mature TGF-beta1 to form the small latent complex. TGF-beta1 binding is predicted to alter LAP conformation, exposing ionic residues (Arg(45), Arg(50), Lys(56), and Arg(58)) on the other side of the alpha-helix, which form the binding site for latent TGF-beta-binding proteins. The stability of the resultant large latent complex is dependent upon covalent dimerization of LAP, which is facilitated by key residues (Phe(198), Asp(199), Val(200), Leu(208), Phe(217), and Leu(219)) at the dimer interface. Significantly, genetic mutations in LAP (e.g. R218H) that cause the rare bone disorder Camurati-Engelmann disease disrupted dimerization and reduced the stability of the latent TGF-beta1 complex.

Tumour necrosis factor-α stimulates human neutrophils to release preformed activin A.

Activin A, a member of the transforming growth factor-ß superfamily, is a critical early mediator of acute inflammation. Activin A release coincides with the release of tumour necrosis factor-α (TNF-α) in models of lipopolysaccharide (LPS)-induced inflammation. The source of circulating activin A during acute inflammation has not been identified and the potential contribution of leukocyte subsets was examined in the following study. Human leukocytes from healthy volunteers were fractionated using Ficoll gradients and cultured under serum-free conditions. Freshly isolated human neutrophils contained 20-fold more activin A than blood mononuclear cells as measured by enzyme-linked immunosorbent assay (ELISA), and both dimeric and monomeric forms of activin A were detected in these cells by western blotting. Activin A was predominantly immunolocalized in the neutrophil cytoplasm. Purified neutrophils secreted activin A in culture when stimulated by TNF-α, but were unable to respond to LPS directly. Although TNF-α stimulated activin A release from neutrophils within 1 h, activin subunit mRNA expression did not increase until 12 h of culture, and the amount of activin A released following TNF-α stimulation did not change between 1 and 12 h. Specific inhibition of the p38 MAP kinase signalling pathway blocked TNF-α-induced activin release, and the secretion of activin A was not due to TNF-α-induced neutrophil apoptosis. These data provide the first evidence that neutrophils are a significant source of mature, stored activin A. Stimulation of the release of neutrophil activin A by TNF-α may contribute to the early peak in circulating activin A levels during acute inflammation.

Expression of nodal signalling components in cycling human endometrium and in endometrial cancer.

BACKGROUND: The human endometrium is unique in its capacity to remodel constantly throughout adult reproductive life. Although the processes of tissue damage and breakdown in the endometrium have been well studied, little is known of how endometrial regeneration is achieved after menstruation. Nodal, a member of the transforming growth factor-beta superfamily, regulates the processes of pattern formation and differentiation that occur during early embryo development. METHODS: In this study, the expression of Nodal, Cripto (co-receptor) and Lefty A (antagonist) was examined by RT-PCR and immunohistochemistry across the menstrual cycle and in endometrial carcinomas. RESULTS: Nodal and Cripto were found to be expressed at high levels in both stromal and epithelial cells during the proliferative phase of the menstrual cycle. Although immunoreactivity for both proteins in surface and glandular epithelium was maintained at relatively steady-state levels across the cycle, their expression was significantly decreased within the stromal compartment by the mid-secretory phase. Lefty expression, as has previously been reported, was primarily restricted to glandular epithelium and surrounding stroma during the late secretory and menstrual phases. In line with recent studies that have shown that Nodal pathway activity is upregulated in many human cancers, we found that Nodal and Cripto immunoreactivity increased dramatically in the transition from histologic Grade 1 to histologic Grades 2 and 3 endometrial carcinomas. Strikingly, Lefty expression was low or absent in all cancer tissues. CONCLUSION: The expression of Nodal in normal and malignant endometrial cells that lack Lefty strongly supports an important role for this embryonic morphogen in the tissue remodelling events that occur across the menstrual cycle and in tumourogenesis.

The transgenerational effects of oocyte mitochondrial supplementation.

Many women suffer from either failed fertilisation or their embryos arrest early during development. Autologous mitochondrial supplementation has been proposed as an assisted reproductive technology to overcome these problems. However, its safety remains to be tested in an animal model to determine if there are transgenerational effects. We have supplemented oocytes with autologous populations of mitochondria to generate founders. We mated the female founders and their offspring to produce three generations. We assessed litter size, the ovarian reserve, and weight gain and conducted a full histopathological analysis from each of the three generations. Across the generations, we observed significant increases in litter size and in the number of primordial follicles in the ovary matched by changes in global gene expression patterns for these early-stage oocytes. However, full histopathological analysis revealed that cardiac structure was compromised in first and second generation offspring, which could seriously affect the health of the offspring. Furthermore, the offspring were prone to increased weight gain during early life. Mitochondrial supplementation appears to perturb the regulation of the chromosomal genome resulting in transgenerational phenotypic gains and losses. These data highlight the need for caution when using autologous mitochondrial supplementation to treat female factor infertility.

Inhibin A and B in vitro bioactivities are modified by their degree of glycosylation and their affinities to betaglycan.

Inhibin A and B, important regulators of normal function in tissues of the reproductive axis, are glycosylated at either Asn(268) or Asn(268) and Asn(302) in the alpha-subunit to produce 31- and 34-kDa isoforms, respectively. In this study, glycosylated isoforms of recombinant human inhibin A and B were purified from conditioned medium using immunoaffinity chromatography and reversed-phase HPLC. The masses of the purified inhibin preparations were determined by several inhibin immunoassays, and their in vitro bioactivities were based on suppression of FSH release by rat pituitary cells in culture. Based on a ratio of in vitro bioactivity to immunoactivity (B:I ratio), the monoglycosylated 31-kDa inhibin A was 5-fold more potent than the diglycosylated 34-kDa inhibin A (B:I ratio, 1.22 +/- 0.15 vs. 0.24 +/- 0.05; P < 0.001, respectively). The 31-kDa inhibin B was significantly (P < 0.001) more potent (1.75 +/- 0.29) than the 34-kDa form (1.08 +/- 0.20). Because inhibin biological activity is dependent upon interactions with the coreceptor betaglycan, the effect of inhibin glycosylation on betaglycan binding was assessed. Analogous to the pattern of in vitro bioactivity, 31-kDa inhibin A was 12-fold more active (IC(50), 0.68 nM) than the 34-kDa isoform (IC(50), 8.2 nM) at displacing [(125)I]inhibin A from COS7 cells expressing betaglycan. However, the 1.6-fold difference in bioactivity of the inhibin B isoforms was not matched by differences in their affinities for betaglycan. It is concluded that glycosylation of Asn(302) of the alpha-subunit of inhibin A and B results in a decrease in bioactivity, and the effect on inhibin A, at least, is explained by its reduced affinity to betaglycan.

Biological activity and in vivo half-life of pro-activin A in male rats.

Mature TGF-ß proteins are used in vivo to promote bone growth, combat obesity, reverse fibrosis and pulmonary arterial hypertension, and as potential rejuvenation factors. However, the serum half-life of this family of growth factors is short (∼5 min), limiting their therapeutic potential. Because TGF-ß proteins are normally secreted from cells with their prodomains attached, we considered whether these molecules could extend the in vivo half-life and activity of their respective growth factors. Using activin A as a model ligand, we initially modified the cleavage site between the pro- and mature domains to ensure complete processing of the activin A precursor. Co-immunoprecipitation studies confirmed mature activin A is secreted from cells in a non-covalent complex with its prodomain, however, the affinity of this interaction is not sufficient to suppress activin A in vitro biological activity. The plasma clearance profiles of purified pro- and mature activin A were determined over a 4 h period in adult male rats. Both activin forms demonstrated a two-phase decay, with the half-life of pro-activin A (t1/2 fast = 12.5 min, slow = 31.0 min) being greater than that of mature activin A (t1/2 fast = 5.5 min, slow = 20.3 min). Both pro- and mature activin A induced significant increases in serum follicle stimulating hormone levels after 4 h, but no differences were observed in the relative in vivo bioactivities of the two activin isoforms. Increased serum half-life of activin A in the presence of its prodomain identifies a new means to increase the therapeutic effectiveness of TGF-ß proteins.

Restoration of normal embryogenesis by mitochondrial supplementation in pig oocytes exhibiting mitochondrial DNA deficiency.

An increasing number of women fail to achieve pregnancy due to either failed fertilization or embryo arrest during preimplantation development. This often results from decreased oocyte quality. Indeed, reduced mitochondrial DNA copy number (mitochondrial DNA deficiency) may disrupt oocyte quality in some women. To overcome mitochondrial DNA deficiency, whilst maintaining genetic identity, we supplemented pig oocytes selected for mitochondrial DNA deficiency, reduced cytoplasmic maturation and lower developmental competence, with autologous populations of mitochondrial isolate at fertilization. Supplementation increased development to blastocyst, the final stage of preimplantation development, and promoted mitochondrial DNA replication prior to embryonic genome activation in mitochondrial DNA deficient oocytes but not in oocytes with normal levels of mitochondrial DNA. Blastocysts exhibited transcriptome profiles more closely resembling those of blastocysts from developmentally competent oocytes. Furthermore, mitochondrial supplementation reduced gene expression patterns associated with metabolic disorders that were identified in blastocysts from mitochondrial DNA deficient oocytes. These results demonstrate the importance of the oocyte's mitochondrial DNA investment in fertilization outcome and subsequent embryo development to mitochondrial DNA deficient oocytes.

Virtual High-Throughput Screening To Identify Novel Activin Antagonists.

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

Inhibin at 90: from discovery to clinical application, a historical review.

When it was initially discovered in 1923, inhibin was characterized as a hypophysiotropic hormone that acts on pituitary cells to regulate pituitary hormone secretion. Ninety years later, what we know about inhibin stretches far beyond its well-established capacity to inhibit activin signaling and suppress pituitary FSH production. Inhibin is one of the major reproductive hormones involved in the regulation of folliculogenesis and steroidogenesis. Although the physiological role of inhibin as an activin antagonist in other organ systems is not as well defined as it is in the pituitary-gonadal axis, inhibin also modulates biological processes in other organs through paracrine, autocrine, and/or endocrine mechanisms. Inhibin and components of its signaling pathway are expressed in many organs. Diagnostically, inhibin is used for prenatal screening of Down syndrome as part of the quadruple test and as a biochemical marker in the assessment of ovarian reserve. In this review, we provide a comprehensive summary of our current understanding of the biological role of inhibin, its relationship with activin, its signaling mechanisms, and its potential value as a diagnostic marker for reproductive function and pregnancy-associated conditions.