Search

Aims: Human relaxin-2 influences renal and cardiovascular functions. We investigated its effects on experimental endothelial dysfunction., Methods and Results: Acetylcholine-mediated vasodilation of rat aortic rings, impaired by 48 h tumour necrosis factor-α (TNF-α) treatment, was dose-dependently improved by relaxin co-incubation, an effect sensitive to phosphatidylinositol 3-kinase (PI3K) inhibition and the glucocorticoid receptor (GR) antagonist RU-486. TNF increased endothelial nitric oxide synthase (eNOS) phosphorylation at Thr495 and decreased total eNOS expression and both basal and stimulated eNOS activity. Relaxin co-incubation did not affect eNOS expression but improved its activity via PI3K-dependent Thr495 dephosphorylation and Ser1177 phosphorylation, and additional Ser633 phosphorylation. Via GR, relaxin attenuated the TNF-related stimulation of endothelin-1 expression, superoxide and nitrotyrosine formation, and arginase II expression. Relaxin restored, via GR-CCAAT/enhancer-binding protein-β (c/EBP-β)-mediated promoter stimulation, the compromised expression of superoxide dismutase-1 (SOD1). In rat aortic endothelial cells, relaxin activated protein kinase B (Akt) and repressed TNF-induced nuclear factor-κB and activator protein-1. Finally, the relevance of the different findings to the model used was proved by pharmacological interventions., Conclusion: Relaxin improved endothelial dysfunction by promoting eNOS activity, suppressing endothelin-1 and arginase-II expression, and up-regulating SOD1 via GR, GR-c/EBP-β, and PI3K-Akt pathways. This corroborates the notion that it functions as an endogenous and potentially therapeutic vasoprotector.

Background: Relaxin-2, a candidate drug for acute heart failure, has been tested successfully in the first human trials. We investigated relaxin's inotropic effects in human myocardium., Methods and Results: In atrial samples from donor (n = 7) and failing (n = 7) hearts, relaxin-2 evoked remarkable positive inotropic effects: showing a half maximum effective concentration of < 1 nmol/L, the maximum peak developed tension (PDT) rose to approximately 270% of baseline, without differences between failing and nonfailing myocardium. The effects critically depended on protein kinase A activation and inhibition of the transient potassium outward current; phosphoinositide-3 kinase inhibition and pertussis toxin pretreatment moderately blunted the effects in nonfailing but markedly suppressed them in failing myocardium. Action potential recordings revealed identical effects of inhibition of the transient potassium outward current and relaxin. In ventricular myocardium, however, relaxin did not show any inotropic effects. The expression of the RXFP1 receptor was moderately decreased in failing compared with nonfailing atrial myocardium but not detectable in any ventricular samples., Conclusions: Relaxin is a positive inotrope in nonfailing and failing human atria, with critical involvement of protein kinase A and inhibition of the transient potassium outward current and an increasing role for G(i) protein-phosphoinositide-3 kinase signaling in failing myocardium., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Increased expression of endothelial adhesion molecules, high levels of the monocyte chemoattractant protein-1 (MCP-1) and enhanced VLA4 integrin/VCAM-1 and CCR-2/MCP-1 interactions are initial steps in vascular inflammation. We sought to determine whether relaxin, a potent vasodilatory and anti-fibrotic agent, mitigates these early events compromising endothelial integrity. The effect of relaxin coincubation on the TNF-α-stimulated expression of the adhesion molecules VCAM-1, ICAM-1 and E-selectin; the MCP-1 expression by human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (HAoSMC); as well as on direct monocyte-endothelium cell adhesion was quantified by ELISA or adhesion assay. CCR-2 and PECAM expression on HUVEC and THP-1 monocytes was investigated by FACS analysis. Relaxin treatment suppressed significantly TNF-α-induced upregulation of VCAM-1 and PECAM, CCR-2, and MCP-1 levels and direct monocyte adhesion to HUVEC. Our findings identify relaxin as a promising inhibitory factor in early vascular inflammation. By attenuating the upregulation of VCAM-1, key adhesion molecule in early vascular inflammation, and of MCP-1, a chemokine pivotal to monocyte recruitment, relaxin decreased initial monocyte-endothelium contact. This may be of relevance for the prevention and treatment of atherosclerosis and of other pro-inflammatory states., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Relaxin peptides act in brain, reproductive and cardiovascular systems, kidneys, and connective tissue through different G protein-coupled receptors. We reported that human relaxin-2 and porcine relaxin are both agonists at the human glucocorticoid receptor (GR). Here, we investigated the possible auto-regulation of relaxin-2 gene expression via recently discovered GR-binding sites in the relaxin-2 promoter. We found that porcine relaxin increased the secretion of human relaxin-like immunoreactivity in HeLa and THP-1 cells. Silencing of GR gene expression completely abolished this effect whereas transfection of wild-type GR into naturally GR-devoid HT-29 cells established relaxin sensitivity. Relaxin was shown to stimulate CAT expression driven by different deletion constructs of the 5'-flanking region of the relaxin-2 promoter. In chromatin immunoprecipitation assays, we detected both GR and relaxin binding to the relaxin-2 promoter. Gel shift assays indicated binding of relaxin-activated GR to half-GREs located between 160 and 200 bp upstream of transcription start but not to the GRE at -900 bp. Relaxin bound to human GR and displaced established GR agonists. Immunofluorescence experiments visualized nuclear co-localization of relaxin and GR in response to relaxin. In conclusion, we have identified a positive auto-regulatory loop of human relaxin-2 expression which involves GR and relaxin/GR binding to half-GREs in the relaxin-2 promoter.

The relaxin peptide family regulates diverse biological functions (central nervous processes, reproduction, cardiovascular and kidney function, and connective tissue composition) through different G protein-coupled receptors. We reported earlier that human relaxin-2 and porcine relaxin additionally interact with the human glucocorticoid receptor (GR) in an agonistic manner. Here we investigated whether the membrane receptor RXFP1 is critically involved in this pathway. We used chemically modified porcine relaxin which was biologically inactive at RXFP1. Native porcine relaxin, but not the modified peptide affected RXFP1-dependent and GR-independent readouts: ERK-1/2 and Akt phosphorylation as well as up-regulation of Akt and endothelin type-B receptor. In contrast, relaxin and modified relaxin inhibited endotoxin-stimulated secretion of TNF-alpha and IL-6 by human macrophages, an effect sensitive to the glucocorticoid receptor antagonists RU-486 and D-06. Both relaxins caused Ser(211) phosphorylation of GR, a biomarker of agonist-related receptor activation. Relaxin-induced accumulation of Ser(211)-phosphorylated GR was found in the cytoplasm and nucleus of HeLa cells, in endothelial cells, and in transfected HT-29 cells. In AP-1-luciferase assays, relaxin and modified relaxin inhibited endotoxin-induced activation of AP-1, a transcription factor essentially involved in endotoxin signaling. This study suggests that the inability of relaxin to interact with its membrane receptor does not interfere with its ability to activate GR.

Relaxin is upregulated and plays a compensatory role in human heart failure. We therefore determined the safety of and dose response to human relaxin in stable patients with heart failure. Sixteen patients were treated with open-label intravenous relaxin in three sequential dose cohorts and monitored hemodynamically during the 24-h infusion and postinfusion periods. The safety demonstrated in group A (treatment for 8 h each with dosages equivalent to 10, 30, and 100 microg/kg/day) allowed escalation to group B (240, 480, and 960 microg/kg/day), and the highest safe dose, 960 microg/kg/day, was selected for a 24-h dosing in group C. Relaxin showed no relevant adverse effects and produced hemodynamic effects consistent with systemic vasodilation, i.e., trends toward increases in the cardiac index and decreases in pulmonary wedge pressure, without inducing hypotension. The first therapeutic use of relaxin in human heart failure demonstrated favorable hemodynamic effects and indicated that it may be of value in the treatment of this widespread disease.

Background: Relaxin is upregulated in human heart failure (HF). Animal and clinical data suggest beneficial hemodynamic and renal effects from vasodilation. We determined safety, tolerability, and pharmacodynamic effects of human Relaxin in stable HF., Methods and Results: Sixteen patients were treated with open-label intravenous Relaxin in 3 dose-escalation cohorts and monitored hemodynamically for 24-hour infusion and postinfusion periods and followed until Day 30. The safety demonstrated in Group A (8-hour sequential infusions at dose levels of 10, then 30, and then 100 microg x kg x day equivalents) allowed escalation to Group B (240, 480, and 960 microg x kg x day). The highest safe dose, 960 microg x kg x day, was selected for a 24-hour infusion in Group C. Relaxin showed no adverse effects; produced hemodynamic effects consistent with vasodilation (ie, trends toward increases in cardiac index, decreases in pulmonary wedge pressure, and decreases in circulating NT-pro BNP without inducing hypotension; improved markers of renal function [creatinine, blood urea nitrogen]). The highest dose caused a transient elevation in creatinine and blood urea nitrogen at Day 9 that was without apparent clinical significance., Conclusions: Relaxin was safe and well-tolerated in patients with stable HF, and preliminary pharmacodynamic responses suggest it causes vasodilation. Further evaluation of the safety and efficacy of this drug in HF appears warranted.

The insulin-related peptide hormone relaxin (Rlx) is known as pregnancy hormone for decades. In the 1980s, researchers began to recognize the highly intriguing fact that Rlx plays a role in a multitude of physiological processes far beyond pregnancy and reproduction. So, Rlx's contribution to the regulation of vasotonus, plasma osmolality, angiogenesis, collagen turnover, and renal function has been established. In addition, the peptide has been demonstrated to represent a mediator of cardiovascular pathology. The ongoing efforts to identify Rlx receptors eventually precipitated the discovery of the G protein-coupled receptors (GPCR) LGR7 and LGR8 as membrane receptors for human Rlx-2 in 2002. This review will summarize the current state of insight into this rapidly evolving field, which has further been expanded by the discovery of GPCR135 and GPCR142 as receptors for Rlx-3. In addition, Rlx has also been shown to activate the human glucocorticoid receptor (GR). There is evidence from Rlx and Rlx receptor knockouts suggesting that LGR7 is the only relevant receptor for mouse Rlx-1 (corresponding to human Rlx-2) in vivo and that insulin-like peptide (INSL)-3 represents the physiological ligand for LGR8. Regarding Rlx signal transduction, the cyclic adenosine monophosphate (cAMP) and nitric oxide (NO) pathways will be characterized as major cascades. Investigation of downstream signaling remains an important field for future research. Finally, the current state of therapeutical strategies using Rlx in animal models as well as in humans is summarized.

The insulin-like peptide relaxin is a central hormone of pregnancy, but it also produces antifibrotic, myocardial, renal, central nervous, and vascular effects. Recently, two G-protein-coupled receptors, LGR7 and LGR8, were identified as relaxin receptors. Prompted by reports on the immunoregulatory effects of relaxin, we investigated possible interactions with the human glucocorticoid receptor (GR). Relaxin blunted the endotoxin-induced production of inflammatory cytokines (interleukin 1 [IL-1], IL-6, and tumor necrosis factor- alpha) by human macrophages, an effect that was suppressed by the GR antagonist RU-486. In three different cell lines, relaxin induced GR activation, nuclear translocation, and DNA binding as assessed in glucocorticoid response element (GRE)-luciferase assays. Coimmunoprecipitation experiments revealed physical interaction of endogenous and exogenous relaxin with cytoplasmic and nuclear GR. Relaxin competed with GR agonists for GR binding both in vivo, in whole-cell assays, and in vitro, in fluorescence polarization assays. In LGR7- and LGR8-free cells, the relaxin-mediated activation of GR was preserved. In conclusion, relaxin acts as a GR agonist, a pathway pivotal to relaxin's effects on cytokine secretion by human macrophages. These findings may deepen our understanding of relaxin's many physiologic actions as well as our insights into general principles of hormone signaling.

Current findings in male relaxin knockout mice indicate antifibrotic and positive lusitropic actions of relaxin on the myocardium. We investigated in 12-month-old male spontaneously hypertensive rats (SHR) and age-matched male Wistar-Kyoto rats (WKY) whether RLX shows antihypertrophic actions as well. SHR showed left heart hypertrophy and a selective elevation of left atrial and ventricular relaxin peptide which inversely correlated with the degree of hypertrophy. In adult rat cardiomyocytes, relaxin blunted phenylephrine-induced hypertrophy by inhibiting activated ERK-1/2 kinases. In conclusion, endogenous myocardial relaxin, upregulated in left heart hypertrophy, exerts antihypertrophic effects by inhibition of activated ERK-1/2 kinases.

The insulin-like peptide relaxin is a central hormone of pregnancy, but it also produces anti-fibrotic, myocardial, renal, central-nervous, and vascular effects. Recently, two G protein-coupled receptors, LGR7 and LGR8, have been identified as relaxin receptors. Prompted by reports on immunoregulatory effects of relaxin, we investigated possible interactions with the human glucocorticoid receptor (GR). Relaxin blunted the endotoxin-induced production of inflammatory cytokines (IL-1, IL-6, TNF-alpha) by human macrophages--an effect that was suppressed by the GR antagonist RU-486. In three different cell lines, relaxin induced GR activation, nuclear translocation, and DNA binding as assessed in GRE-luciferase assays. Co-immunoprecipitation experiments revealed physical interaction of endogenous and exogenous relaxin with cytoplasmic and nuclear GR. Relaxin competed with GR agonists for GR binding, both in vivo in whole-cell assays, and in vitro in fluorescence polarization assays. Relaxin was shown to up-regulate GR protein expression as well as the number of functionally active GR sites. In LGR7/8-free cells, the relaxin-mediated activation of GR was preserved. In conclusion, relaxin acts as GR agonist--a pathway pivotal to its effects on cytokine secretion by human macrophages. These findings may deepen our understanding of relaxin's abundant physiological actions, as well as our insights into general principles of hormone signaling.

We have recently demonstrated that relaxin (RLX) acts as compensatory mediator in human heart failure. RLX inhibits the stimulation of endothelin-1, the most potent vasoconstrictor in heart failure. Upregulation of the endothelin type-B receptor (ET(B)), which mediates endothelin-1 clearance and endothelial release of NO, represents a pivotal mode of RLX action. However, signal transduction and abundance of this phenomenon are unknown. Therefore, we investigated RLX-induced regulation of ET(B) in human umbilical vein endothelial, epithelial (HeLa), and vascular smooth muscle cells. In human umbilical vein endothelial cells and HeLa cells, but not in human vascular smooth muscle cells, RLX upregulated ET(B) expression and activated extracellular signal-regulated kinase-1/2 (ERK-1/2) and nuclear factor-kappaB (NF-kappaB), a transcription factor. PD-98059, a selective inhibitor of the mitogen-activated protein kinase kinase-1 (MEK-1)-ERK-1/2 pathway, abolished ERK-1/2 and NF-kappaB activation and ET(B) upregulation. NF-kappaB inhibition also prevented RLX-mediated ET(B) stimulation. In NF-kappaB-luciferase reporter assays, we demonstrated complete inhibition of RLX-induced NF-kappaB activation in cells transfected with dominant-negative Raf-1, MEK-1, or ERK-1/2 constructs, whereas dominant-negative Ras had no effect. In rat aorta and mesenteric artery, RLX pretreatment, in an ET(B)-dependent fashion, mitigated the maximum contractile response to endothelin-1, by 38+/-4% and 43+/-6%, and the endothelin-1 sensitivity (-log[EC(50)]: aorta, 8.2+/-0.2 for vehicle versus 7.2+/-0.2 for RLX; mesenteric artery, 8.0+/-0.2 for vehicle versus 7.1+/-0.1 for RLX). RLX pretreatment augmented the dilator effect of the ET(B) agonist endothelin-3 by 100+/-8% and 133+/-13%. In conclusion, RLX stimulates endothelial and epithelial ET(B) via a Ras-independent Raf-1-MEK-1-ERK-1/2 pathway that activates NF-kappaB. On vascular smooth muscle cells, ET(B), a contributor to endothelin-mediated vasoconstriction, remains unaffected. This renders RLX a functional endothelin-1 antagonist.