Cornelia Bartsch

The pregnancy hormone relaxin is a player in human heart failure

Human congestive heart failure is characterized by complex neurohumoral activation associated with the up‐regulation of vasoconstricting and salt‐retaining mediators and the compensatory rise of counter‐regulatory hormones. In the present study, we provide the first evidence that relaxin (RLX), known as a pregnancy hormone, represents a potential compensatory mediator in human heart failure: plasma concentrations of RLX and myocardial expression of the two RLX genes (H1 and H2) correlate with the severity of disease and RLX responds to therapy. The failing human heart is a relevant source of circulating RLX peptides, and myocytes as well as interstitial cells produce RLX. Elevation of ventricular filling pressure up‐regulates RLX expression and the hormone acts as a potent inhibitor of endothelin 1, the most powerful vasoconstrictor in heart failure. Furthermore, RLX modulates effects of angiotensin II, another crucial mediator. Our data identify RLX as a new player in human heart failure with potential diagnostic and therapeutic relevance.—Dschietzig, T., Richter, C., Bartsch, C., Laule, M., Armbruster, F. P., Baumann, G., Stangl, K. The pregnancy hormone relaxin is a player in human heart failure. FASEB J. 15, 2187–2195 (2001)

Relaxin, a pregnancy hormone, is a functional endothelin-1 antagonist: Attenuation of endothelin-1-mediated vasoconstriction by stimulation of endothelin type-B receptor expression via ERK-1/2 and nuclear factor-κb

Abstract— 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 (ETB), 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 ETB 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 ETB expression and activated extracellular signal–regulated kinase-1/2 (ERK-1/2) and nuclear factor-&kgr;B (NF-&kgr;B), 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-&kgr;B activation and ETB upregulation. NF-&kgr;B inhibition also prevented RLX-mediated ETB stimulation. In NF-&kgr;B-luciferase reporter assays, we demonstrated complete inhibition of RLX-induced NF-&kgr;B 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 ETB-dependent fashion, mitigated the maximum contractile response to endothelin-1, by 38±4% and 43±6%, and the endothelin-1 sensitivity (−log[EC50]: 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 ETB agonist endothelin-3 by 100±8% and 133±13%. In conclusion, RLX stimulates endothelial and epithelial ETB via a Ras-independent Raf-1–MEK-1–ERK-1/2 pathway that activates NF-&kgr;B. On vascular smooth muscle cells, ETB, a contributor to endothelin-mediated vasoconstriction, remains unaffected. This renders RLX a functional endothelin-1 antagonist.

Identification of the pregnancy hormone relaxin as glucocorticoid receptor agonist

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‐α) 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.

Plasma levels and cardiovascular gene expression of urotensin-II in human heart failure

The peptide urotensin-II (U-II) has been described as most potent vasoconstrictor identified so far, but plasma values in humans and its role in cardiovascular pathophysiology are unknown. We investigated circulating urotensin-II and its potential role in human congestive heart failure (CHF). We enrolled control individuals (n=13; cardiac index [CI], 3.5+/-0.1 l/min/m2; pulmonary wedge pressure [PCWP], 10+/-1 mm Hg), patients with moderate (n=10; CI, 2.9+/-0.3 l/min/m2; PCWP, 14+/-2 mm Hg) and severe CHF (n=11; CI, 1.8+/-0.2 l/min/m2; PCWP, 33+/-2 mm Hg). Plasma levels of urotensin-II differed neither between controls, patients with moderate and severe CHF nor between different sites of measurement (pulmonary artery, left ventricle, coronary sinus, antecubital vein) within the single groups. Hemodynamic improvement by vasodilator therapy in severe CHF (CI, +78+/-3%; PCWP, -55+/-3%) did not affect circulating U-II over 24 h. Preprourotensin-II mRNA expression in right atria, left ventricles, mammary arteries and saphenous veins did not differ between controls with normal heart function and patients with end-stage CHF. In conclusion, urotensin-II plasma levels and its myocardial and vascular gene expression are unchanged in human CHF. Circulating urotensin-II does not respond to acute hemodynamic improvement. These findings suggest that urotensin-II does not play a major role in human CHF.

Relaxin improves TNF-α-induced endothelial dysfunction: the role of glucocorticoid receptor and phosphatidylinositol 3-kinase signalling.

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.

Relaxin inhibits early steps in vascular inflammation.

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.

Myocardial Relaxin Counteracts Hypertrophy in Hypertensive Rats

Abstract: 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.

RXFP1-inactive relaxin activates human glucocorticoid receptor: Further investigations into the relaxin–GR pathway

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.

The adrenomedullin receptor acts as clearance receptor in pulmonary circulation

Adrenomedullin (AM) is a powerful pulmonary vasodilator with antimitogenic properties. We investigated the role of the AM receptor (AMR) and the calcitonin gene-related peptide type-1 receptor (CGRP1R) in regulating pulmonary vascular AM levels. The AMR antagonist hAM(22-52) (120 nmol/L) significantly elevated AM release compared with controls to 250% after 2 h in isolated rat lungs and to 830% after 4 h in pulmonary artery endothelial cells (PAEC). CGRP1R blockade had no effect. AMR blockade did not influence prepro-AM mRNA levels nor did inhibition of protein synthesis by cycloheximide (0.01 mg/mL) abolish the effect of the AMR antagonist. Radioligand-binding studies with PAEC membranes revealed a decrease by 44% of the AMR density in response to AMR antagonism. Altogether, the pulmonary vascular AMR represents not only a functionally active, but also a clearance receptor; its expression is constitutively stimulated by basal AM. This identifies a novel mechanism for controlling pulmonary AM levels.

The Pregnancy Hormone Relaxin Binds to and Activates the Human Glucocorticoid Receptor

Abstract: 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‐ α) 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 relaxins effects on cytokine secretion by human macrophages. These findings may deepen our understanding of relaxins many physiologic actions as well as our insights into general principles of hormone signaling.