Targeting the relaxin-3/RXFP3 system: a patent review for the last two decades.

INTRODUCTION: The neuropeptide relaxin-3/RXFP3 system belongs to the relaxin/insulin superfamily and is involved in many important physiological processes, such as stress responses, appetite control, and motivation for reward. Although relaxin-3 is the endogenous agonist for RXFP3, it can also bind to and activate RXFP1 and RXFP4. Consequently, research has been focused on the development of RXFP3-specific peptides and small-molecule ligands to validate the relaxin-3/RXFP3 system as a novel drug target. AREAS COVERED: This review provides an overview of patents on the relaxin-3/RXFP3 system covering ligand development and pharmacological studies since 2003. Related patents and literature reports were obtained from established sources including SciFinder, Google Patents, and Espacenet for patents and SciFinder, PubMed, and Google Scholar for literature reports. EXPERT OPINION: There has been an increasing amount of patent activities around relaxin-3/RXFP3, highlighting the importance of this novel neuropeptide system for drug discovery. The development of relaxin-3 derived peptides and small-molecule modulators, as well as behavioral studies in rodents, have shown that the relaxin-3/RXFP3 system is a promising drug target for treating various metabolic and neuropsychiatric diseases including obesity, anxiety, and alcohol addiction.

The consequences of manipulating relaxin family peptide receptor 1 (RXFP1) level in ovarian cancer cells.

Deregulation of the relaxin family peptide system (RFPS) appears to increase the risk of range of cancers, including epithelial ovarian cancers (EOC). The present study examines the effect of relaxin family peptide receptor 1 (RXFP1) level on the biological properties of human epithelial ovarian adenocarcinoma cells (OVCAR4 and SKOV3). RXFP1 was downregulated (RXFP1↓) in the cells using the RXFP1 sgRNA CRISPR All-in-One Lentivirus set (pLenti-U6-sgRNA-SFFV-Cas9-2A-Puro), and upregulated (RXFP1↑) using the RXFP1 CRISPRa sgRNA Lentivector (pLenti-U6-sgRNA-PGK-Neo) kit, which activates the RXFP1 gene when paired with dCas9-SAM. The changes taking place during adhesion to extracellular matrix (ECM) proteins were assessed in multi-well plates coated with collagen, fibronectin, laminin and gelatin. Cellular viability was monitored based on mitochondrial metabolic activity (MTT Assay, Alamar Blue Assay) and adenosine triphosphate production (ATP Assay). The rate of cell proliferation was determined based on the percentage of Ki67 immunoreactive cells and the numbers of cells in particular cell-cycle phases. The mesenchymal-like (Boyden Chamber Assay) and amoeboid-like movements (Wound Healing Assay) of ovarian cancer cells were also analyzed after transfection. RXFP1 downregulation decreased the adhesion properties of ovarian cancer cells and increased the tendency for apoptosis under stressful conditions. In contrast, RXFP1 upregulation had pro-proliferative, pro-survival and promigratory effects. Our findings confirm that the relaxin-2/RXFP1 signaling pathway plays a role in the promotion of growth and progression of ovarian cancer.

Total Chemical Synthesis of Aggregation-Prone Disulfide-Rich Starfish Peptides.

: A relaxin-like gonad-stimulating peptide (RGP), Aso-RGP, featuring six cysteine residues, was identified in the Crown-of-Thorns Starfish (COTS, Acanthaster cf. solaris) and initially produced through recombinant yeast expression. This method yielded a single-chain peptide with an uncleaved C-peptide (His Tag) and suboptimal purity. Our objective was to chemically synthesize Aso-RGP in its mature form, comprising two chains (A and B) and three disulfide bridges, omitting the C-peptide. Furthermore, we aimed to synthesize a newly identified relaxin-like peptide, Aso-RLP2, from COTS, which had not been previously synthesized. This paper report the first total chemical synthesis of Aso-RGP and Aso-RLP2. Aso-RGP synthesis proceeded without major issues, whereas the A-chain of Aso-RLP2, in its reduced and unfolded state with two free thiols, presented considerable challenges. These were initially marked by "messy" RP-HPLC profiles, typically indicative of synthesis failure. Surprisingly, oxidizing the A-chain significantly improved the RP-HPLC profile, revealing the main issue was not synthesis failure but the peptide's aggregation tendency, which initially obscured analysis. This discovery highlights the critical need to account for aggregation in peptide synthesis and analysis. Ultimately, our efforts led to the successful synthesis of both peptides with purities exceeding 95%.

Knee Laxities Changes with Sex-steroids throughout the Menstrual Cycle Phases in Athlete and Non-athlete Females.

Objective: Our study investigated changes of knee laxities in athletes and non-athletes females and relationship between knee laxity and sex-steroid at menstrual cycle phases. Methods: Forty six healthy females, twenty four athletes and twenty two non-athletes not on hormone contraceptive pills, had no previous knee injuries and with regular menstrual cycles for 3 consecutive months, participated in the study. Medial and lateral knee laxities were determined by varus-valgus tests at follicular, ovulatory and luteal phases. Serum level of relaxin, estrogen, progesterone and testosterone were determined by ELISA and radioimmunoassay. Results: Knee laxities in athletes and non-athletes at 0° and 20° flexion were the highest in luteal phase with non-athletes possess greater laxity than athletes. Positive correlation between progesterone and relaxin levels with knee laxities were observed. Meanwhile, the levels of both hormones were highest in the luteal phase. Conclusion: Increased medial and lateral knee laxities in athletes and non-athletes associated with high serum progesterone and relaxin levels in luteal phase may contribute toward increased risk of non-contact knee injury. However, lower knee laxity in athletes than non-athletes suggest that exercise could be a protective factor.

Pathologic maternal and neonatal outcomes associated with programmed embryo transfer: potential etiologies and strategies for prevention.

PURPOSE: In the first of two companion papers, we comprehensively reviewed the recent evidence in the primary literature, which addressed the increased prevalence of hypertensive disorders of pregnancy, late-onset or term preeclampsia, fetal overgrowth, postterm birth, and placenta accreta in women conceiving by in vitro fertilization. The preponderance of evidence implicated frozen embryo transfer cycles and, specifically, those employing programmed endometrial preparations, in the higher risk for these adverse maternal and neonatal pregnancy outcomes. Based upon this critical appraisal of the primary literature, we formulate potential etiologies and suggest strategies for prevention in the second article. METHODS: Comprehensive review of primary literature. RESULTS: Presupposing significant overlap of these apparently diverse pathological pregnancy outcomes within subjects who conceive by programmed autologous FET cycles, shared etiologies may be at play. One plausible but clearly provocative explanation is that aberrant decidualization arising from suboptimal endometrial preparation causes greater than normal trophoblast invasion and myometrial spiral artery remodeling. Thus, overly robust placentation produces larger placentas and fetuses that, in turn, lead to overcrowding of villi within the confines of the uterine cavity which encroach upon intervillous spaces precipitating placental ischemia, oxidative and syncytiotrophoblast stress, and, ultimately, late-onset or term preeclampsia. The absence of circulating corpus luteal factors like relaxin in most programmed cycles might further compromise decidualization and exacerbate the maternal endothelial response to deleterious circulating placental products like soluble fms-like tyrosine kinase-1 that mediate disease manifestations. An alternative, but not mutually exclusive, determinant might be a thinner endometrium frequently associated with programmed endometrial preparations, which could conspire with dysregulated decidualization to elicit greater than normal trophoblast invasion and myometrial spiral artery remodeling. In extreme cases, placenta accreta could conceivably arise. Though lower uterine artery resistance and pulsatility indices observed during early pregnancy in programmed embryo transfer cycles are consistent with this initiating event, quantitative analyses of trophoblast invasion and myometrial spiral artery remodeling required to validate the hypothesis have not yet been conducted. CONCLUSIONS: Endometrial preparation that is not optimal, absent circulating corpus luteal factors, or a combination thereof are attractive etiologies; however, the requisite investigations to prove them have yet to be undertaken. Presuming that in ongoing RCTs, some or all adverse pregnancy outcomes associated with programmed autologous FET are circumvented or mitigated by employing natural or stimulated cycles instead, then for women who can conceive using these regimens, they would be preferable. For the 15% or so of women who require programmed FET, additional research as suggested in this review is needed to elucidate the responsible mechanisms and develop preventative strategies.

Musculoskeletal Morphology and Joint Flexibility-Associated Functional Characteristics across Three Time Points during the Menstrual Cycle in Female Contemporary Dancers.

Findings are inconsistent with regards to whether menstrual cycle phase-associated changes in physical functioning exist. It is possible that such discrepancies are due to varying rigour in experimental approaches. The current study aimed to systematically evaluate any effect of carefully tracked menstrual cycle phase on precisely measured muscle structure and function in a physically active group (contemporary dancers). Eleven women aged (M [SD]) 23.5 [2.94] years, undergoing 10.5 [1.73] hours of contemporary dance practice and 6.12 [2.36] hours of other physical activity per week, were recruited. Sex hormone level (enzyme-linked immunosorbent assays (ELISA), skin temperature and ovulation kits), physical pain assessments (Ice Water Test, Visual Analogue Scale, The Physical Activity Readiness Questionnaire, Self-Estimated Functional Inability Because of Pain Questionnaire, and Pain Anxiety Symptoms Scale), muscle architecture measurement (B-mode ultrasonography), and physical functioning (dynamometry, force-platform and electromyography) on both lower limbs were measured at three time points during one cycle, following three months of menstrual cycle monitoring. There was no difference in musculoskeletal flexibility variables between follicular, ovulatory, or luteal phases. Nonetheless, oestrogen change was associated with variability in 11 musculoskeletal variables, progesterone change was associated with variability in 7, and relaxin change was associated with variability in 15. Negative correlations existed between progesterone and flexibility and between oestrogen and jump variables. Moreover, oestrogen and relaxin were associated with increased musculoskeletal compliance, whilst progesterone was associated with increased muscle stiffness. In short, in absolute sex hormone levels, 'inter-individual' variances appear more impactful than 'intra-individual' variances. Not only are oestrogen and progesterone associated with differing musculoskeletal outcomes, but relaxin is also associated with musculoskeletal compliance changes. These effects are anticipated to impact jump height and flexibility, and hence, they could be expected to affect overall physical performance, including dance.

The involvement of the Wnt/ß-catenin signaling cascade in fibrosis progression and its therapeutic targeting by relaxin.

Organ scarring, referred to as fibrosis, results from a failed wound-healing response to chronic tissue injury and is characterised by the aberrant accumulation of various extracellular matrix (ECM) components. Once established, fibrosis is recognised as a hallmark of stiffened and dysfunctional tissues, hence, various fibrosis-related diseases collectively contribute to high morbidity and mortality in developed countries. Despite this, these diseases are ineffectively treated by currently-available medications. The pro-fibrotic cytokine, transforming growth factor (TGF)-ß1, has emerged as the master regulator of fibrosis progression, owing to its ability to promote various factors and processes that facilitate rapid ECM synthesis and deposition, whilst negating ECM degradation. TGF-ß1 signal transduction is tightly controlled by canonical (Smad-dependent) and non-canonical (MAP kinase- and Rho-associated protein kinase-dependent) intracellular protein activity, whereas its pro-fibrotic actions can also be facilitated by the Wnt/ß-catenin pathway. This review outlines the pathological sequence of events and contributing roles of TGF-ß1 in the progression of fibrosis, and how the Wnt/ß-catenin pathway contributes to tissue repair in acute disease settings, but to fibrosis and related tissue dysfunction in synergy with TGF-ß1 in chronic diseases. It also outlines the anti-fibrotic and related signal transduction mechanisms of the hormone, relaxin, that are mediated via its negative modulation of TGF-ß1 and Wnt/ß-catenin signaling, but through the promotion of Wnt/ß-catenin activity in acute disease settings. Collectively, this highlights that the crosstalk between TGF-ß1 signal transduction and the Wnt/ß-catenin cascade may provide a therapeutic target that can be exploited to broadly treat and reverse established fibrosis.

R2R01: A long-acting single-chain peptide agonist of RXFP1 for renal and cardiovascular diseases.

BACKGROUND: The therapeutic potential of relaxin for heart failure and renal disease in clinical trials is hampered by the short half-life of serelaxin. Optimization of fatty acid-acetylated single-chain peptide analogues of relaxin culminated in the design and synthesis of R2R01, a potent and selective RXFP1 agonist with subcutaneous bioavailability and extended half-life. EXPERIMENTAL APPROACH: Cellular assays and pharmacological models of RXFP1 activation were used to validate the potency and selectivity of R2R01. Increased renal blood flow was used as a translational marker of R2R01 activity. Human mastocytes (LAD2 cells) were used to study potential pseudo-allergic reactions and CD4+ T-cells to study immunogenicity. The pharmacokinetics of R2R01 were characterized in rats and minipigs. KEY RESULTS: In vitro, R2R01 had comparable potency and efficacy to relaxin as an agonist for human RXFP1. In vivo, subcutaneous administration of R2R01 increased heart rate and renal blood flow in normotensive and hypertensive rat and did not show evidence of tachyphylaxis. R2R01 also increased nipple length in rats, used as a chronic model of RXFP1 engagement. Pharmacokinetic studies showed that R2R01 has a significantly extended terminal half-life. The in vitro assays with LAD2 cells and CD4+ T-cells showed that R2R01 had low potential for pseudo-allergic and immunogenic reactions, respectively. CONCLUSION AND IMPLICATIONS: R2R01 is a potent RXFP1 agonist with an extended half-life that increases renal blood flow in various settings including normotensive and hypertensive conditions. The preclinical efficacy and safety data supported clinical development of R2R01 as a potential new therapy for renal and cardiovascular diseases.

Evolution of the human birth canal.

It seems puzzling why humans have evolved such a small and rigid birth canal that entails a relatively complex process of labor compared with the birth canal of our closest relatives, the great apes. This study reviewed insights into the evolution of the human birth canal from recent theoretical and empirical studies and discussed connections to obstetrics, gynecology, and orthopedics. Originating from the evolution of bipedality and the large human brain million years ago, the evolution of the human birth canal has been characterized by complex trade-off dynamics among multiple biological, environmental, and sociocultural factors. The long-held notion that a wider pelvis has not evolved because it would be disadvantageous for bipedal locomotion has not yet been empirically verified. However, recent clinical and biomechanical studies suggest that a larger birth canal would compromise pelvic floor stability and increase the risk of incontinence and pelvic organ prolapse. Several mammals have neonates that are equally large or even larger than human neonates compared to the size of the maternal birth canal. In these species, the pubic symphysis opens widely to allow successful delivery. Biomechanical and developmental constraints imposed by bipedality have hindered this evolutionary solution in humans and led to the comparatively rigid pelvic girdle in pregnant women. Mathematical models have shown why the evolutionary compromise to these antagonistic selective factors inevitably involves a certain rate of fetopelvic disproportion. In addition, these models predict that cesarean deliveries have disrupted the evolutionary equilibrium and led to new and ongoing evolutionary changes. Different forms of assisted birth have existed since the stone age and have become an integral part of human reproduction. Paradoxically, by buffering selection, they may also have hindered the evolution of a larger birth canal. Many of the biological, environmental, and sociocultural factors that have influenced the evolution of the human birth canal vary globally and are subject to ongoing transitions. These differences may have contributed to the global variation in the form of the birth canal and the difficulty of labor, and they likely continue to change human reproductive anatomy.

Methods for the induction of labor: efficacy and safety.

This review assessed the efficacy and safety of pharmacologic agents (prostaglandins, oxytocin, mifepristone, hyaluronidase, and nitric oxide donors) and mechanical methods (single- and double-balloon catheters, laminaria, membrane stripping, and amniotomy) and those generally considered under the rubric of complementary medicine (castor oil, nipple stimulation, sexual intercourse, herbal medicine, and acupuncture). A substantial body of published reports, including 2 large network meta-analyses, support the safety and efficacy of misoprostol (PGE1) when used for cervical ripening and labor induction. Misoprostol administered vaginally at doses of 50 µg has the highest probability of achieving vaginal delivery within 24 hours. Regardless of dosing, route, and schedule of administration, when used for cervical ripening and labor induction, prostaglandin E2 seems to have similar efficacy in decreasing cesarean delivery rates. Globally, although oxytocin represents the most widely used pharmacologic agent for labor induction, its effectiveness is highly dependent on parity and cervical status. Oxytocin is more effective than expectant management in inducing labor, and the efficacy of oxytocin is enhanced when combined with amniotomy. However, prostaglandins administered vaginally or intracervically are more effective in inducing labor than oxytocin. A single 200-mg oral tablet of mifepristone seems to represent the lowest effective dose for cervical ripening. The bulk of the literature assessing relaxin suggests this agent has limited benefit when used for this indication. Although intracervical injection of hyaluronidase may cause cervical ripening, the need for intracervical administration has limited the use of this agent. Concerning the vaginal administration of nitric oxide donors, including isosorbide mononitrate, isosorbide, nitroglycerin, and sodium nitroprusside, the higher incidence of side effects with these agents has limited their use. A synthetic hygroscopic cervical dilator has been found to be effective for preinduction cervical ripening. Although a pharmacologic agent may be administered after the use of the synthetic hygroscopic dilator, in an attempt to reduce the interval to vaginal delivery, concomitant use of mechanical and pharmacologic methods is being explored. Combining the use of a single-balloon catheter with dinoprostone, misoprostol, or oxytocin enhances the efficacy of these pharmacologic agents in cervical ripening and labor induction. The efficacy of single- and double-balloon catheters in cervical ripening and labor induction seems similar. To date, the combination of misoprostol with an intracervical catheter seems to be the best approach when balancing delivery times with safety. Although complementary methods are occasionally used by patients, given the lack of data documenting their efficacy and safety, these methods are rarely used in hospital settings.

Maternal Fed Zinc-Deficient Diet: Effects on Relaxin Family Peptides and Oxidant System in the Testis and Liver Tissue of Male Offspring.

Today, the studies are limited on roles of insulin-like peptide 3 (INSL3), insulin-like peptide 7 (INSL7), and relaxin family peptide receptor 1 (RXFP1) which are synthesized by the testis. It is aimed to investigate the levels of the sex hormone as testosterone and the family of insulin-like proteins (relaxin family peptides), which are important in the puberty transition, in the testicular and liver tissues of male offspring born to female rats fed a zinc-deficient diet during the pregnancy, and in the changes in lipid peroxidation markers. The study was performed on 40 male offspring. In Group I: Control group, both male offspring and mothers were fed with standard rat chow. In Group II: Zinc deficient diet, both male offspring and mothers were fed a zinc-deficient diet (2.8 mg/kg zinc). In Group III: Normal diet, male offspring fed standard rat chow for 45 days (66th day) after being separated from their mothers with a maternal zinc-deficient diet. In Group IV: Zinc-supplemented diet, offspring fed with zinc supplemented (5 mg/kg/day intraperitoneal zinc sulfate, i.p.) in addition to standard rat chow after being separated from their mothers with maternal zinc deficiency until the termination of the study (66th day). Our study suggests that zinc-supplemented diets play an important role in the changes in INSL3, INSL7, RXFP1, and testosterone levels during spermatogenesis. INSL7, INSL3, and RXFP1 levels were higher in zinc-supplemented group than the zinc-deficient diet group. Liver levels of INSL3, INSL7, and MDA were significantly different in zinc-deficiency diet group than zinc-supplemented group.

Expression of Relaxin Family Peptide Receptors 1 and 3 in the Ovarian Follicle of Japanese Quail.

In our previous studies, we demonstrated that the primary source of relaxin 3 (RLN3) in Japanese quail is ovarian granulosa cells. Although several relaxin family peptide (RXFP) receptors have been sequenced, the intricacies of these receptors in avian species remain insufficiently clarified. Therefore, we assessed the expression of RXFP receptors, RXFP1 and 3, in Japanese quail. Using RT-PCR, we found that both RXFP1 and 3 were ubiquitously expressed. The expression level of RXFP1 is significantly higher in the ovarian theca layer, indicating that it is the primary receptor for RLN3 in the ovary. During follicular development, there was an elevation in thecal RXFP1 expression, but it declined after the luteinizing hormone (LH) surge. We found that the protease activity of the 60 kDa band increased after the LH surge, suggesting the involvement of RLN3 signaling in ovulation. These results suggest a paracrine role of RLN3, involving its binding with RXFP1 in ovarian theca cells. This interaction may elicit biological actions, potentially initiating ovulation after the LH surge.

The diverse influences of relaxin-like peptide family on tumor progression: Potential opportunities and emerging challenges.

Relaxin-like peptide family exhibit differential expression patterns in various types of cancers and play a role in cancer development. This family participates in tumorigenic processes encompassing proliferation, migration, invasion, tumor microenvironment, immune microenvironment, and anti-cancer resistance, ultimately influencing patient prognosis. In this review, we explore the mechanisms underlying the interaction between the RLN-like peptide family and tumors and provide an overview of therapeutic approaches utilizing this interaction.

Programmed Death-Ligand (PD-L1), Epidermal Growth Factor (EGF), Relaxin, and Matrix Metalloproteinase-3 (MMP3): Potential Biomarkers of Malignancy in Canine Mammary Neoplasia.

Gene expression has been suggested as a putative tool for prognosis and diagnosis in canine mammary neoplasia (CMNs). In the present study, 58 formalin-fixed paraffin-embedded (FFPE) paraffined canine mammary neoplasias from 27 different bitches were included. Thirty-seven tumours were classified as benign, whereas thirty-one were classified as different types of canine carcinoma. In addition, mammary samples from three healthy bitches were also included. The gene expression for vascular endothelial growth factor-α (VEGFα), CD20, progesterone receptor (PGR), hyaluronidase-1 (HYAL-1), programmed death-ligand 1 (PD-L1), epidermal growth factor (EGF), relaxin (RLN2), and matrix metalloproteinase-3 (MMP3) was assessed through RT-qPCR. All the assessed genes yielded a higher expression in neoplastic mammary tissue than in healthy tissue. All the evaluated genes were overexpressed in neoplastic mammary tissue, suggesting a role in the process of tumorigenesis. Moreover, PD-L1, EGF, relaxin, and MMP3 were significantly overexpressed in malignant CMNs compared to benign CMNs, suggesting they may be useful as malignancy biomarkers.

Molecular mechanisms of Chengshi Beixie Fenqing Decoction based on network pharmacology: pivotal roles of relaxin signaling pathway and its associated target proteins against Benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is a common disease that affects the quality of life of middle-aged and older men. We investigated the therapeutical effects of Chengshi Beixie Fenqing Decoction (CBFD), a classic traditional Chinese medicine prescription, on BPH through in vivo model and network pharmacology. Bioactives in CBFD were detected through UPLC-Q-Tof-MS/MS and GC-MS, and filtered by the modified Lipinski's rule. Target proteins associated with the filtered compounds and BPH are selected from public databases. Venn diagram identified the overlapping target proteins between the bioactives-interacted target proteins and the BPH-targeted proteins. The bioactive-protein interactive networking of BPH was analyzed through the KEGG pathway on STRING to identify potential ligand-target and visualized the rich factors on the R packet. After that, the molecular docking test (MDT) was performed between bioactives and target proteins. It showed that the mechanism of CBFD against BPH was related to 104 signaling pathways of 42 compounds. AKT1, 6-demethyl-4'-methyl-N-methylcoclaurine and relaxin signaling pathways were selected as a hub target, key bioactivitie and hub signaling pathway, respectively. In addition, three major compounds, 6-demethyl-4'-methyl-N-methylcoclaurine, isoliensinine and liensinine, had the highest affinity on MDT for the three crucial target proteins, AKT1, JUN and MAPK1. These proteins were associated with the relaxin signaling pathway, which regulated the level of nitric oxide and is implicated in both BPH development and CBFD. We concluded that the three key bioactivities found in Plumula nelumbinis of CBFD may contribute to improving BPH condition by activating the relaxin signaling pathways.Communicated by Ramaswamy H. Sarma.

Specific expression and blood kinetics for relaxin 2, lipocalin 2, and tissue factor pathway inhibitor 2 at the canine placenta and pregnant bloods.

In general, humoral factors released from the placenta influence pregnancy progression, but the involvement of the canine placenta is often unidentified. We investigated specific genes in canine placentas and analyzed the blood dynamics of the translated proteins. Furthermore, RNAs are known to be released from placentas embedding in exosomes, a type of extracellular vesicles. Here, the presence of cell-free RNAs in pregnant serums was also confirmed. RNA specimens were purified from the normal healthy dog placentas and applied to RNA-Seq analysis. Expressions of frequent genes were confirmed by RT-PCR using placentas from other individuals and breeds. Relaxin (RLN) 2, lipocalin (LCN) 2, and tissue factor pathway inhibitor (TFPI) 2 were selected as high-expressed and placenta-specific genes. By western blot, the three factors were clearly detected in the pregnant serums. Quantitative analysis revealed that the amount of RLN2 increased significantly from non-pregnancy to day 41 of pregnancy. Regarding LCN2 and TFPI2, the protein serum levels elevated during pregnancy, but the statistical differences were not detected. Exosomes were found in all pregnant serums; however, the percentage was less than 6% in total extracellular vesicles. The cell-free RNA related to RLN2 was detected, but no elevation was confirmed during pregnancy. We found specific genes in the canine placenta and the transition of their translated protein into the blood. These factors may become useful tools for research on canine pregnancy and monitoring of reproductive management. Exosomes and cell-free RNA could not be found to be valid in canine reproduction.

Parental History of Hypertension: A Risk for Autonomic Dysfunction and Metabolic and Vascular Derangement in Normotensive Male Offspring.

BACKGROUND: Children of hypertensive parents have an increased propensity of developing hypertension, at an age very much prior to their parents. Understanding the pathophysiology of hypertension in such young individuals, especially baroreflex sensitivity (BRS), is necessary. Reduced heart rate variability (HRV), insulin resistance (IR), dyslipidemia, and decreased vasodilatory adipokines, namely, apelin and relaxin, in normotensives may predispose to the onset of hypertension. Thus, this study compared autonomic functions, vascular markers, and metabolic profiles between normotensive male offspring with and without parental hypertension. METHODS: This analytical cross-sectional study comprised 40 male normotensive offspring of hypertensive parents, aged 18-35 years, recruited as the study group and 40 age- and body mass index (BMI)-matched normotensive male offspring with non-hypertensive parents enrolled as controls. Cardiovascular autonomic functions, including BRS, HRV, diastolic blood pressure response to isometric handgrip test (ΔDBPIHG), Valsalva ratio, and metabolic and vascular markers, were assessed. RESULTS: The study group exhibited reduced BRS, HRV, and Valsalva ratio and higher ΔDBPIHG compared to controls, indicating impaired autonomic functions. The study group had higher IR and triglyceride levels and reduced apelin and relaxin levels. BRS showed significant correlations with HRV, Valsalva ratio, ΔDBPIHG, and metabolic and vascular markers. CONCLUSIONS: Normotensive male offspring of hypertensive parents exhibit impaired autonomic functions, as evidenced by reduced BRS, HRV, and Valsalva ratio. Additionally, they have higher IR, dyslipidemia, and decreased levels of vasodilatory adipokines, indicating an increased risk for future hypertension development. These findings signify that early identification of hypertensive potential in this high-risk population is warranted, which would enable taking necessary preventive measures.

Relaxin treatment stimulates the differentiation of mesenchymal stem cells into osteoblasts.

Background/purpose: Several studies have determined that relaxin stimulates differentiation and regulates the activity of mature osteoclasts, but little is known about its effect on the differentiation of mesenchymal cells towards the osteogenic lineage. Therefore, this study aimed to determine the effect of relaxin on the proliferation and differentiation of the osteoblastic lineage of mesenchymal cells derived from human dental pulp (hDPSC). Materials and methods: In this in vitro study, hDPSC were characterized and treated with relaxin at different doses (10-80 ng/ml) and times (1-21 days). Morphology was assessed by microscopy, and proliferation was assessed using a resazurin assay. Osteoblastic differentiation was evaluated by Alizarin Red staining, alkaline phosphatase (ALP) labeling, and changes in the expression of the osteoblastic differentiation genes RUNX2 and BMP2. Results: Relaxin treatment did not induce changes in the proliferation or viability of hDPSCs; however, larger cells and increased cytoplasmic prolongation were observed. Relaxin treatment (20 and 80 ng/ml) significantly increased calcified nodule formation on days 14 and 21. The cytochemical signals for ALP, RUNX2, and BMP2 gene expression were significantly (P < 0.05) increased by the relaxin treatment. Conclusion: Relaxin treatment does not induce changes in hDPSC proliferation but induces morphological changes, increases ALP detection, calcified nodule formation, and increases expression of RUNX2 and BMP2, suggesting the induction of osteoblastic differentiation of hDPSC.

Expression of Relaxin 3 in the Ovarian Follicle of Japanese Quail.

The relaxin (RLN) gene is expressed in the reproductive tracts, such as the ovary and uterus, of mammalian species. Although RLN expression is detected in the chicken ovary, detailed clarification of the physiological role of RLN has not yet been reported. To address this issue, in the present study we aimed to examine the spatiotemporal expression and hormonal control of RLN in Japanese quail. By performing semi-quantitative and quantitative reverse transcription-polymerase chain reaction analysis, we found that RLN mRNA was mainly expressed in the granulosa and theca layers of the ovary. The expression level in the granulosa layer increased with the stage of follicular development. Results from granulosa layer culture experiments revealed that RLN mRNA expression increased with the addition of estradiol-17ß, whereas the addition of progesterone suppressed RLN transcription. More detailed analysis indicated that RLN expression was highest in the stigma region of the follicle but significantly decreased as the time of the expected luteinizing hormone (LH) surge approached. Together, our findings demonstrated that the granulosa cells in the mature preovulatory follicles constitute the main source of RLN in the Japanese quail. Because RLN expression was highest in the stigma region and the expression dramatically decreased following the LH surge, the results further suggest that RLN may be related to tissue remodeling for the ovulation process in birds.

Novel RXFP3 negative allosteric modulator RLX-33 reduces alcohol self-administration in rats.

There is much interest in identifying novel pharmacotherapeutic targets that improve clinical outcomes for the treatment of alcohol use disorder (AUD). One promising target for therapeutic intervention is the relaxin family peptide 3 (RXFP3) receptor, a cognate receptor for neuropeptide relaxin-3, which has previously been implicated in regulating alcohol drinking behavior. Recently, we developed the first small-molecule RXFP3-selective negative allosteric modulator (NAM) RLX-33. Therefore, the goal of the present work was to characterize the impact of this novel NAM on affective-related behaviors and alcohol self-administration in rats. First, the effects of RLX-33 were tested on alcohol and sucrose self-administration in Wistar and alcohol-preferring P rats to determine the dose-response profile and specificity for alcohol. Then, we assessed the effects of systemic RLX-33 injection in Wistar rats in a battery of behavioral assays (open-field test, elevated zero maze, acoustic startle response test, and prepulse inhibition) and tested for alcohol clearance. We found that the lowest effective dose (5 mg/kg) reduced alcohol self-administration in both male and female Wistar rats, while in alcohol-preferring P rats, this effect was restricted to males, and there were no effects on sucrose self-administration or general locomotor activity. The characterization of affective and metabolic effects in Wistar rats generally found few locomotor, affective, or alcohol clearance changes, particularly at the 5 mg/kg dose. Overall, these findings are promising and suggest that RXFP3 NAM has potential as a pharmacological target for treating AUD.