Further Developments towards a Minimal Potent Derivative of Human Relaxin-2.

Human relaxin-2 (H2 relaxin) is a peptide hormone with potent vasodilatory and anti-fibrotic effects, which is of interest for the treatment of heart failure and fibrosis. H2 relaxin binds to the Relaxin Family Peptide Receptor 1 (RXFP1). Native H2 relaxin is a two-chain, three-disulfide-bond-containing peptide, which is unstable in human serum and difficult to synthesize efficiently. In 2016, our group developed B7-33, a single-chain peptide derived from the B-chain of H2 relaxin. B7-33 demonstrated poor affinity and potency in HEK cells overexpressing RXFP1; however, it displayed equivalent potency to H2 relaxin in fibroblasts natively expressing RXFP1, where it also demonstrated the anti-fibrotic effects of the native hormone. B7-33 reversed organ fibrosis in numerous pre-clinical animal studies. Here, we detail our efforts towards a minimal H2 relaxin scaffold and attempts to improve scaffold activity through Aib substitution and hydrocarbon stapling to re-create the peptide helicity present in the native H2 relaxin.

Relaxin-2 during pregnancy according to glycemia, continence status, and pelvic floor muscle function.

INTRODUCTION AND HYPOTHESIS: To investigate relaxin-2 concentration comparing gestational diabetes mellitus (GDM) and non-GDM patients during pregnancy according to urinary incontinence (UI) and pelvic function status. METHODS: This is a cross-sectional study evaluating 282 pregnant women from 24 weeks of gestation. The participants were divided into two groups, non-GDM and GDM, according to American Diabetes Association's diabetes mellitus gestational threshold. In addition, according to subanalysis, both groups were subdivided according to the presence of pregnancy-specific urinary incontinence: non-GDM continent, non-GDM incontinent, GDM continent, and GDM incontinent. All participants filled in questionnaires on clinical, obstetric, and urinary continence status (International Consultation on Incontinence Questionnaire-Short Form, ICIQ-SF, and Incontinence Severity Index, ISI), followed by pelvic floor muscle evaluation by the PERFECT scheme in which strength, endurance, and speed of contractions were evaluated. RESULTS: Serum relaxin-2 concentrations were significantly lower in pregnant women with pregnancy-specific urinary incontinence in both non-GDM and GDM patients, but GDM showed the lowest concentration. In addition, the stratification of the groups according to pelvic floor muscle strength showed that pregnant patients with GDM and modified Oxford scale 0-2 had significantly lower levels than those who were non-GDM and GDM with Modified Oxford Scale 3-5. Relaxin-2 level was much lower in GDM incontinent pregnant women with MOS 0-2 compared to the other three groups. CONCLUSIONS: Lower relaxin-2 concentration was associated with the presence of pregnancy-specific urinary incontinence, but the combination of GDM, pregnancy-specific urinary incontinence, and lower levels of pelvic floor strength led to lower levels of relaxin-2 compared to the other three groups.

Advancement in female sports medicine and preventive medicine.

Sport plays a major role in maintaining and improving physical function and health of women from adolescence to sexual maturity, through to menopause and old age, and it also plays a key role in social productivity and medicine from the perspective of preventive medicine. However, in routine clinical practice, there are many gynecological problems that affect the condition and performance of female athletes due to a lack of appropriate medical intervention, such as neglect of amenorrhea and menstruation-related symptoms. In addition, the number of athletes aiming to return to competition post-partum has been increasing in recent years, but there is little medical data on the well-being of female athletes during pregnancy and the post-partum period. I previously conducted clinical research on three separate topics, with the aim of clarifying the current issues unique to female athletes, mainly in terms of conditioning and injury prevention. The goal was that clinical research was to examine the situation of athletes with and without disabilities and provide feedback from the survey results to athletes and coaches. This paper divides the clinical studies conducted to date into three topics that will be explored herein: (i) research on the female athlete triad (Triad); (ii) research on oral contraceptives/low-dose estrogen-progestin; and (iii) research on pregnancy and post-partum period.

Rh-relaxin-2 attenuates degranulation of mast cells by inhibiting NF-κB through PI3K-AKT/TNFAIP3 pathway in an experimental germinal matrix hemorrhage rat model.

BACKGROUND: Mast cells play an important role in early immune reactions in the brain by degranulation and the consequent inflammatory response. Our aim of the study is to investigate the effects of rh-relaxin-2 on mast cells and the underlying mechanisms in a germinal matrix hemorrhage (GMH) rat model. METHODS: One hundred seventy-three P7 rat pups were subjected to GMH by an intraparenchymal injection of bacterial collagenase. Clodronate liposome was administered through intracerebroventricular (i.c.v.) injections 24 h prior to GMH to inhibit microglia. Rh-relaxin-2 was administered intraperitoneally at 1 h and 13 h after GMH. Small interfering RNA of RXFP1 and PI3K inhibitor LY294002 were given by i.c.v. injection. Post-GMH evaluation included neurobehavioral function, Western blot analysis, immunofluorescence, Nissl staining, and toluidine blue staining. RESULTS: Our results demonstrated that endogenous relaxin-2 was downregulated and that RXFP1 level peaked on the first day after GMH. Administration of rh-relaxin-2 improved neurological functions, attenuated degranulation of mast cells and neuroinflammation, and ameliorated post-hemorrhagic hydrocephalus (PHH) after GMH. These effects were associated with RXFP1 activation, increased expression of PI3K, phosphorylated AKT and TNFAIP3, and decreased levels of phosphorylated NF-κB, tryptase, chymase, IL-6, and TNF-α. However, knockdown of RXFP1 and PI3K inhibition abolished the protective effects of rh-relaxin-2. CONCLUSIONS: Our findings showed that rh-relaxin-2 attenuated degranulation of mast cells and neuroinflammation, improved neurological outcomes, and ameliorated hydrocephalus after GMH through RXFP1/PI3K-AKT/TNFAIP3/NF-κB signaling pathway.

Tissue-specific relaxin-2 is differentially associated with the presence/size of an arterial aneurysm and the severity of atherosclerotic disease in humans.

Circulating or tissue-related biomarkers are of clinical value for risk stratification in patients with abdominal aortic aneurysms. Relaxin-2 (RL2) has been linked to the presence and size of arterial aneurysms, and to the extent of atherosclerosis in human subjects. Here, we assessed the expression levels of RL2 in aneurysmal (AA, n = 16) and atherosclerotic (ATH, n = 22) arteries, and established the correlation between RL2 levels and the presence/size of AA and the clinical severity of atherosclerosis. The expression levels of metalloproteinases (MMPs) and endothelial nitric oxide synthetase (eNOS) were also detected for correlations with different phenotypes of atherosclerosis and AA. Temporal artery biopsy specimens (n = 6) and abdominal aortic tissues harvested from accident victims during autopsy (n = 10) were used as controls. Quantitative tissue biomarker analysis revealed that tissue-specific RL2 was increased in patients with larger or symptomatic AA compared to subjects with atherosclerotic disease and healthy controls. In situ RL2 levels were proportional to the size and the severity of aneurysmatic disease, and were substantially elevated in patients with symptomatic aneurysm of any diameter or asymptomatic aneurysm of a diameter >350% of that of the normal artery. In contrast, tissue RL2 was inversely associated with the clinical severity of atherosclerotic lesions. Correlation between RL2 and MMP2 was different between ATH1 and ATH2, depending on atherosclerosis grade. Overall, tissue RL2 is differentially associated with discrete phenotypes of arterial disease and might exert multipotent biological effects on vascular wall integrity and remodeling in human subjects.

Altered Cerebral Blood Flow and Potential Neuroprotective Effect of Human Relaxin-2 (Serelaxin) During Hypoxia or Severe Hypovolemia in a Sheep Model.

Specific neuroprotective strategies to minimize cerebral damage caused by severe hypoxia or hypovolemia are lacking. Based on previous studies showing that relaxin-2/serelaxin increases cortical cerebral blood flow, we postulated that serelaxin might provide a neuroprotective effect. Therefore, we tested serelaxin in two emergency models: hypoxia was induced via inhalation of 5% oxygen and 95% nitrogen for 12 min; thereafter, the animals were reoxygenated. Hypovolemia was induced and maintained for 20 min by removal of 50% of the total blood volume; thereafter, the animals were retransfused. In each damage model, the serelaxin group received an intravenous injection of 30 µg/kg of serelaxin in saline, while control animals received saline only. Blood gases, shock index values, heart frequency, blood pressure, and renal blood flow showed almost no significant differences between control and treatment groups in both settings. However, serelaxin significantly blunted the increase of lactate during hypovolemia. Serelaxin treatment resulted in significantly elevated cortical cerebral blood flow (CBF) in both damage models, compared with the respective control groups. Measurements of the neuroproteins S100B and neuron-specific enolase in cerebrospinal fluid revealed a neuroprotective effect of serelaxin treatment in both hypoxic and hypovolemic animals, whereas in control animals, neuroproteins increased during the experiment. Western blotting showed the expression of relaxin receptors and indicated region-specific differences in relaxin receptor-mediated signaling in cortical and subcortical brain arterioles, respectively. Our findings support the hypothesis that serelaxin is a potential neuroprotectant during hypoxia and hypovolemia. Due to its preferential improvement of cortical CBF, serelaxin might reduce cognitive impairments associated with these emergencies.

Receptor-independent modulation of TGF-ß-induced pro-fibrotic pathways by relaxin-2 in human primary tubular epithelial cells.

Renal tubular epithelial cells actively contribute to the development of renal fibrosis and may be targeted by anti-fibrotic drugs. Relaxin-2 (RLX2) applied as recombinant protein is suggested to be renoprotective. Therefore, we investigated whether human primary tubular epithelial cells (hPTEC) obtained from various donors were target cells for the anti-fibrotic actions of RLX2. Treatment of hPTEC with RLX2 reduced the TGF-ß1-induced secretion of the pro-fibrotic factor CTGF (connective tissue growth factor) and inhibited fibronectin synthesis and secretion. Furthermore, metalloproteinase MMP2 secretion was increased, with no effect on MMP9. Considerable differences were observed between hPTEC obtained from different donors. Therefore, expression of the relaxin family peptide receptor RXFP1, the major mediator of renal RLX2 effects, was analyzed. A validated antibody detected a double band of 80-90 kDa in cellular homogenates by Western blotting. Expression of the detected protein was not altered by incubation with TGF-ß1 and RLX2-induced modulation of CTGF expression did not correlate with the putative receptor expression. Therefore, relaxin family receptors RXFP1-4 were assessed by RNA-seq analysis. No evidence was found for mRNA expression of any of these receptors in several hPTEC preparations. Lack of RXFP1 mRNA was confirmed by qPCR using mRNA obtained from THP-1 cells as positive control. Our data thus provide evidence for primary renal human tubular epithelial cells as targets for the anti-fibrotic actions of RLX2. However, anti-fibrotic effects were observed at micromolar concentrations of RLX2 and shown to be independent of RXFP1 expression.

The subset of patients with acute heart failure able to secrete relaxin-2 at pregnancy concentrations could have a longer survival: a pilot study.

OBJECTIVE: To investigate how many patients with acute heart failure (AHF) hypersecrete relaxin-2 concentrations similar to those of pregnant women and determine their long-term outcome. METHODS: In consecutive AHF patients relaxin-2 was quantified by ELISA sandwich method. Patients were divided into pregnancy-like group (PLG, relaxin-2 ≥ 500 pg/mL) and control group (CG, relaxin-2 < 500 pg/mL). The primary outcome was all-cause death during follow-up. Secondary endpoints were prolonged hospitalisation (>10 days), combined endpoint (death, rehospitalisation, ED revisit) 30 days after discharge, and 30-day, one-year and three-year death rates. RESULTS: We included 814 patients [81 (SD = 9) years; 53.0% women] followed during 1.9 (SD 2.8) years; 517 (63.5%) died. Twenty patients (2.5%) formed the PLG (median relaxin-2 = 1459 pg/mL; IQR = 1722) and 794 the CG (median = 26; IQR = 44). There was no interaction with variables included on adjustment (age, sex, ischaemic cardiomyopathy, NT-proBNP, glycaemia, and sodium). PLG patients did not have better short-term secondary endpoints, but did show a significantly lower three-year mortality [ORadjusted = 0.17 (0.05-0.5), p = 0.003]. CONCLUSIONS: The small proportion of AHF patients achieving relaxin-2 concentrations similar to those observed in pregnancy may survive longer.

Relaxin-2 therapy reverses radiation-induced fibrosis and restores bladder function in mice.

AIM: To determine the efficacy of human relaxin-2 (hRLX2) in reversing radiation-induced bladder fibrosis and lower urinary tract dysfunction (LUTD). Radiation cystitis is a consequence of radiotherapy for pelvic malignancies. Acutely, irradiation leads to reactive oxygen/nitrogen species in urothelial cells, apoptosis, barrier disruption, and inflammation. Chronically, this results in collagen deposition, bladder fibrosis, and attenuated storage and voiding functions. In severe cases, cystectomies are performed as current therapies do not reverse fibrosis. METHODS: We developed a mouse model for selective bladder irradiation (10 Gray; 1 Gy = 100 rads) resulting in chronic fibrosis within 6 weeks, with decreased bladder compliance, contractility, and overflow incontinence. Seven weeks post-irradiation, female C57Bl/6 mice were continuously infused with hRLX2 (400 µg/kg/day/14 days) or vehicle (saline) via subcutaneous osmotic pumps. Mice were evaluated in vivo using urine spot analysis, cystometrograms and external urethral sphincter electromyograms; and in vitro using length-tension measurements, Western blots, histology, and immunohistochemistry. RESULTS: hRLX2 reversed fibrosis, decreased collagen content, improved bladder wall architecture, and increased bladder compliance, detrusor smooth muscle Cav1.2 expression and detrusor contractility in mice with chronic radiation cystitis. hRLX2 treatment outcomes were likely caused by the activation of RXFP1/2 receptors which are expressed on the detrusor. CONCLUSION: hRLX2 may be a new therapeutic option for rescuing bladders with chronic radiation cystitis.

Relaxin 2/RXFP1 Signaling Induces Cell Invasion via the ß-Catenin Pathway in Endometrial Cancer.

Relaxin is known to play an important role in animal pregnancies, including those of humans. It is suggested that relaxin induces aggressive cell growth and invasiveness in several types of cancer, including endometrial cancer. However, the mechanisms of relaxin remain largely unclear. In this study, we examined the effects of relaxin 2 (RLN2), the major circulating relaxin in humans, on human endometrial carcinoma cell lines. RLN2 treatment induced invasion in HEC-1B and Ishikawa cells. RLN2-induced cell invasion was significantly decreased by transfection of relaxin receptor 1 (RXFP1) siRNAs. The ß-catenin inhibitor, XAV939, also significantly inhibited the RLN2-induced cell invasions. Both a decrease of cadherin expression and an increase of ß-catenin phosphorylation were observed in response to the RLN2 treatment in HEC-1B and Ishikawa cells. We then examined RLN2 and RXFP1 expression in 80 human endometrioid endometrial carcinoma tissues. RLN2 immunoreactivity was detected in the human endometrial carcinoma cells and had a correlative tendency with histological grade and RXFP1. These results suggest that adherens junctions in cancer cells are weakened by the breakdown of the cadherin/catenin complex, which is induced by ß-catenin phosphorylation via RLN2/RXFP1 signaling.

Relaxin reduces susceptibility to post-infarct atrial fibrillation in mice due to anti-fibrotic and anti-inflammatory properties.

BACKGROUND: Relaxin-2 (RLX) is a peptide hormone that exerts beneficial anti-fibrotic and anti-inflammatory effects in diverse models of cardiovascular disease. The goal of this study was to determine the effects of RLX treatment on the susceptibility to atrial fibrillation (AF) after myocardial infarction (MI). METHODS: Mice with cryoinfarction of the left anterior ventricular wall were treated for two weeks with either RLX (75 µg/kg/d) or vehicle (sodium acetate) delivered via subcutaneously implanted osmotic minipumps. RESULTS: RLX treatment significantly attenuated the increase in AF-inducibility following cryoinfarction and reduced the mean duration of AF episodes. Furthermore, epicardial mapping of both atria revealed an increase in conduction velocity. In addition to an attenuation of atrial hypertrophy, chronic application of RLX reduced atrial fibrosis, which was linked to a significant reduction in atrial mRNA expression of connective tissue growth factor. Transcript levels of the pro-inflammatory cytokines interleukin-6 and interleukin-1ß were reduced in RLX treated mice, but macrophage infiltration into atrial myocardium was similar in the vehicle and RLX treated groups. CONCLUSION: Treatment with RLX in mice after MI reduces susceptibility to AF due to anti-inflammatory and anti-fibrotic properties. Because to these favorable actions, RLX may become a new therapeutic option in the treatment of AF, even when complicating MI.

Alterations of relaxin and its receptor system components in experimental diabetic cardiomyopathy rats.

High glucose induces apoptosis of cardiomyocytes and fibrosis of cardiac fibroblasts, contributing to diabetic cardiomyopathy. In this work, we explore the production of relaxin alterations and the significance of their receptor system components in the hearts of experimental diabetic cardiomyopathy rats. We measured rat relaxin-1 (equivalent to human relaxin-2), relaxin-3, RXFP1 and RXFP3 mRNA expression in the hearts of experimental diabetic cardiomyopathy rats. Neonatal rat ventricular myocytes (NRVMs) and cardiac fibroblasts were treated with 5.5 mmol/l normal glucose (NG) and 33 mmol/l high glucose (HG) for 0, 6, 12, 24, 48 and 72 h. Rat relaxin-1, relaxin-3, RXFP1 and RXFP3 mRNA expression were determined by real-time PCR. In the present study, we offer the first evidence that Relaxin-1 mRNA significantly increased and Relaxin-3 mRNA expression decreased at 4 and 8 weeks after STZ in the hearts of diabetic rats. In addition, significant down regulation of the mRNA expression of RXFP1 and RXFP3 was observed at 4 w after STZ; however, the mRNA expression levels of RXFP1 and RXFP3 were increased at 8 weeks after STZ. Apoptotic NRVMs induced by high glucose generate a decreased level of relaxin-1 and RXFP1. In HG-administered cardiac fibroblasts, Relaxin-1 mRNA was significantly increased and relaxin-3 mRNA was significantly decreased. Additionally, the mRNA expression of RXFP1 was decreased, and the mRNA expression of RXFP3 was increased. This results showed that an important role of relaxin-2, relaxin-3 and their receptors system in the regulation of diabetic cardiomyopathy.

Imbalanced Angiogenesis in Peripartum Cardiomyopathy　- Diagnostic Value of Placenta Growth Factor.

BACKGROUND: Concentrations of the anti-angiogenic factor soluble fms-like tyrosine kinase-1 (sFlt-1) are altered in peripartum cardiomyopathy (PPCM). In this study we investigated changes in the angiogenesis balance in PPCM.Methods and Results:Plasma concentrations of sFlt-1 and the pro-angiogenic placenta growth factor (PlGF) were determined in patients with PPCM during the post-partum phase (n=83), in healthy women at delivery (n=30), and in patients with acute heart failure (AHF; n=65). Women with cardiac failure prepartum or associated with any form of hypertension, including pre-eclampsia, were excluded. Compared with non-pregnant women, in women with AHF and PPCM, median PlGF concentrations were greater (19 [IQR 16-22] and 98 [IQR 78-126] ng/mL, respectively; P<0.001) and the sFlt-1/PlGF ratio was lower (9.8 [6.6-11.3] and 1.2 [0.9-2.8], respectively; P<0.001). The sFlt-1/PlGF ratio was lower in PPCM than in normal deliveries (1.2 [0.9-2.8] vs. 94.8 [68.8-194.1], respectively; P<0.0001). The area under the curve for PlGF (cut-off value: 50ng/mL) and/or the sFlt-1/PlGF ratio (cut-off value: 4) to distinguish PPCM from either normal delivery or AHF was >0.94. Median plasma concentrations of the anti-angiogenic factor relaxin-2 were lower in PPCM and AHF (0.3 [IQR 0.3-1.7] and 0.3 [IQR 0.3-1] ng/mL, respectively) compared with normal deliveries (1,807 [IQR 1,101-4,050] ng/mL; P<0.001). CONCLUSIONS: Plasma of PPCM patients shows imbalanced angiogenesis. High PlGF and/or low sFlt-1/PlGF may be used to diagnose PPCM.

Opposite actions of urotensin II and relaxin-2 on cellular expression of fibronectin in renal fibrosis: A preliminary experimental study.

Our aim was to evaluate the role of urotensin II, urantide (urotensin II receptor antagonist) and relaxin-2 on the cellular expression of fibronectin as a surrogate marker for renal fibrosis. We employed LLC-PK1 renal tubular epithelial cells and assessed the influence on the fibrotic process of the above-mentioned substances by using anti-fibronectin antibodies in western blot analysis. The addition of urotensin II increased fibronectin expression. Urantide reduced the positivity for fibronectin caused by urotensin II (P<.05). The anti-fibrotic action was more evident for relaxin-2 (P<.01). Also in the model of TGF-ß1-induced fibrosis, urantide and, to a greater extent, relaxin-2 were able to significantly lessen fibronectin expression (respectively, P<.05 and P<.01). In conclusion, relaxin-2 may reduce urotensin II-induced renal fibrosis.

Oral contraceptive therapy reduces serum relaxin-2 in elite female athletes.

AIM: Recent investigations have demonstrated that athletes with high relaxin-2 levels have a high risk of anterior cruciate ligament injuries, while athletes taking oral contraceptives (OC) have low relaxin-2 levels. It has not yet been clarified whether taking OC reduces relaxin-2 levels. The purpose of this study was to investigate changes in relaxin-2 levels in athletes taking OC. METHODS: Levels of relaxin-2, estradiol, progesterone, luteinizing hormone and follicle-stimulating hormone were measured in serum samples (n = 183) from 106 elite female athletes. Five athletes with serum relaxin-2 concentrations > 6 pg/mL during the luteal phase were recruited to assess the effect of OC therapy. RESULTS: Serum relaxin-2 concentrations were significantly higher during the luteal phase (n = 57) than in the follicular phase (n = 72), or in athletes on OC therapy (n = 10) (P < 0.001, P < 0.001 and P < 0.05, respectively). In the luteal phase, 36.8% (21/57) of the athletes had relaxin levels > 6 pg/mL. In 23 athletes, serum relaxin-2 concentrations were measured during both the follicular and luteal phases, revealing that relaxin-2 levels were significantly higher in the luteal phase compared with the follicular phase. In 5 out of 23 athletes, serum relaxin-2 concentrations were > 6 pg/mL in the luteal phase and during the second cycle of OC therapy, relaxin-2 concentrations decreased dramatically to below the detection limit (0.26 pg/mL). CONCLUSIONS: High serum relaxin-2 concentrations were only detected during the luteal phase. In athletes with high relaxin-2 concentrations during the luteal phase, OC therapy decreased serum relaxin-2 levels.

Increase of cortical cerebral blood flow and further cerebral microcirculatory effects of Serelaxin in a sheep model.

Serelaxin, recombinant human relaxin-2, modulates endothelial vasodilatory functionality and is under evaluation for treatment of acute heart failure. Little is known about acute effects on cerebral perfusion. We tested the hypothesis that Serelaxin might also have effects on the cerebral microcirculation in a sheep model, which resembles human brain structure quite well. We used laser Doppler flowmetry and sidestream dark-field (SDF) imaging techniques, which are reliable tools to continuously assess dynamic changes in cerebral perfusion. Laser Doppler flowmetry shows that bolus injection of 30 µg Serelaxin/kg body wt induces an increase (P = 0.006) to roughly 150% of cortical cerebral blood flow (CBF), whereas subcortical CBF remains unchanged (P = 0.688). The effects on area-dependent CBF were significantly different after the bolus injection (P = 0.042). Effects on cortical CBF were further confirmed by SDF imaging. The bolus injection of Serelaxin increased total vessel density to 127% (P = 0.00046), perfused vessel density to 145% (P = 0.024), and perfused capillary density to 153% (P = 0.024). Western blotting confirmed the expression of relaxin receptors RXFP1 and truncated RXFP2-variants in the respective brain regions, suggesting a possible contribution of RXFP1 on the effects of Serelaxin. In conclusion, the injection of a high dose of Serelaxin exerts quick effects on the cerebral microcirculation. Therefore, Serelaxin might be suitable to improve cortical microcirculation and exert neuroprotective effects in clinically relevant scenarios that involve cortical hypoperfusion. These findings need to be confirmed in relevant experimental settings involving cerebral cortical hypoperfusion and can possibly be translated into clinical practice.

Relaxin-2 expression in temporal bone carcinoma.

Temporal bone squamous cell carcinoma (TBSCC) is an uncommon, aggressive malignancy with a significant local recurrence rate even in patients with postoperative pathology reports of free surgical margins. This raises the question of how "free" negative margins should be to be oncologically safe, especially in bone tissue. A potential role for relaxin-2 hormone in tumor-driven osteolysis has recently been reported. The aim of this study was to assess the prognostic role of relaxin-2 expression in TBSCC tissue specimens and pathologically negative bone margins. Relaxin-2 immunohistochemical expression was assessed in 25 consecutively operated TBSCC patients. Several pathological variables correlated with recurrence rate (pT stage, dura mater involvement), disease-free survival (DFS) (pT stage, pN status, grade, and dura mater involvement), and disease-specific survival (DSS) (pT stage, pN status, grade, and dura mater involvement). The recurrence rate, DFS, and DSS did not correlate with relaxin-2 expression in TBSCC specimens or pathologically negative bone margins. Although local recurrence in TBSCC could relate to neoplastic bone invasion not apparent on conventional pathological investigations, the present preliminary findings seem to rule out any role of relaxin-2 in mediating this local aggressiveness. Molecular mechanisms of TBSCC recurrence after curative treatment should be further investigated.

Presence of relaxin-2, oxytocin and their receptors in uterosacral ligaments of pre-menopausal patients with and without pelvic organ prolapse.

OBJECTIVE: Pelvic organ prolapse (POP) is a major health concern for women. Its pathophysiology is yet not fully understood. We reported an impaired functional state of the smooth muscle compartment in uterosacral ligaments from patients with POP, which was cholinergic, stimulated by oxytocin and modulated by relaxin-2. The current study investigated the presence of oxytocin and relaxin-2 and their receptors in the uterosacral ligament from POP/non-POP patients. DESIGN: Translational investigation on clinical samples. SETTING: University hospital departments. POPULATION AND SAMPLES: Fourty-three samples of uterosacral ligament from pre-menopausal patients with (n = 20) and without POP (n = 23). METHODS: Presence of relaxin-2, its receptors RXFP1 and RXFP2, and of oxytocin and its receptor were analysed by immunohistochemistry and classified using a staining score. Additionally, Western blot analysis was performed. MAIN OUTCOME MEASURES: Presence patterns with respect to POP and non-POP uterosacral ligament samples for pathophysiological understanding of POP. RESULTS: Relaxin-2, oxytocin and their receptors were expressed in endothelial cells, the smooth muscle compartment and vasa vasorum in the arteries and veins of the uterosacral ligament, in the smooth muscle compartment present in the ground reticulum and in nerves running through the uterosacral ligament. The presence level of relaxin-2 was higher in the uterosacral ligament of the POP cohort (p < 0.001). CONCLUSIONS: We demonstrated that relaxin-2 had an increased presence in uterosacral ligaments from patients with POP, suggesting a role of the relaxin system in the pathogenesis of POP and identifying the relaxin system as a potential therapeutic target for the pharmacological treatment of POP.

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.

Prognostic association of circulating relaxin-2 in acute heart failure.

BACKGROUND: Despite the increasing interest in the study of the endogenous relaxin system in heart failure (HF), its role as a prognostic marker in acute HF remains unclear. We aimed to evaluate the association of relaxin-2 circulating levels with 6 months' mortality in acute HF. METHODS: We evaluated relaxin-2 serum levels at admission in a cohort of patients with acute HF (n = 202) using an enzyme immunoassay. The ability of relaxin-2 to predict all-cause death (primary outcome) and HF-specific death (secondary outcome) at 6 months was assessed using Cox-regression analysis. RESULTS: The median age was 79 (70-85) years old, 44% of the patients were male, and 43% had preserved ejection fraction (≥50%). Median serum relaxin-2 level was 25 pg/mL. Patients with higher relaxin-2 levels had more peripheral oedemas, higher sodium retention score, higher pulmonary artery pressures, higher prevalence of right ventricle dysfunction and lower inferior vena cava collapse at inspiration. Conversely, there was no association with left chambers parameters or with B-type natriuretic peptide (BNP). Higher relaxin-2 concentrations were associated with a higher risk of all-cause death [HR 1.15; 95%CI 1.01,1.30; P = 0.030] and HF-specific death [HR 1.21; 95% CI 1.03-1.42; P = 0.018], after adjustment for classical prognostic factors such as age, sex and BNP. CONCLUSIONS: In our acute HF population, relaxin-2 circulating levels were associated with clinical and echocardiographic markers of systemic congestion and with 6-months' mortality, independently of BNP. These results lay the groundwork for future investigations on the potential of relaxin-2 as an auxiliary biomarker in HF.