Thomas Dschietzig

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.

Intravenous Recombinant Human Relaxin in Compensated Heart Failure: A Safety, Tolerability, and Pharmacodynamic Trial

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.

Relaxin, a pleiotropic vasodilator for the treatment of heart failure

Relaxin is a naturally occurring peptide hormone that plays a central role in the hemodynamic and renovascular adaptive changes that occur during pregnancy. Triggering similar changes could potentially be beneficial in the treatment of patients with heart failure. The effects of relaxin include the production of nitric oxide, inhibition of endothelin, inhibition of angiotensin II, production of VEGF, and production of matrix metalloproteinases. These effects lead to systemic and renal vasodilation, increased arterial compliance, and other vascular changes. The recognition of this has led to the study of relaxin for the treatment of heart failure. An initial pilot study has shown favorable hemodynamic effects in patients with heart failure, including reduction in ventricular filling pressures and increased cardiac output. The ongoing RELAX-AHF clinical program is designed to evaluate the effects of relaxin on the symptoms and outcomes in a large group of patients admitted to hospital for acute heart failure. This review will summarize both the biology of relaxin and the data supporting its potential efficacy in human heart failure.

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.

International Union of Basic and Clinical Pharmacology. XCV. Recent Advances in the Understanding of the Pharmacology and Biological Roles of Relaxin Family Peptide Receptors 1–4, the Receptors for Relaxin Family Peptides

Relaxin, insulin-like peptide 3 (INSL3), relaxin-3, and INSL5 are the cognate ligands for the relaxin family peptide (RXFP) receptors 1–4, respectively. RXFP1 activates pleiotropic signaling pathways including the signalosome protein complex that facilitates high-sensitivity signaling; coupling to Gαs, Gαi, and Gαo proteins; interaction with glucocorticoid receptors; and the formation of hetero-oligomers with distinctive pharmacological properties. In addition to relaxin-related ligands, RXFP1 is activated by Clq-tumor necrosis factor-related protein 8 and by small-molecular-weight agonists, such as ML290 [2-isopropoxy-N-(2-(3-(trifluoromethylsulfonyl)phenylcarbamoyl)phenyl)benzamide], that act allosterically. RXFP2 activates only the Gαs- and Gαo-coupled pathways. Relaxin-3 is primarily a neuropeptide, and its cognate receptor RXFP3 is a target for the treatment of depression, anxiety, and autism. A variety of peptide agonists, antagonists, biased agonists, and an allosteric modulator target RXFP3. Both RXFP3 and the related RXFP4 couple to Gαi/Gαo proteins. INSL5 has the properties of an incretin; it is secreted from the gut and is orexigenic. The expression of RXFP4 in gut, adipose tissue, and β-islets together with compromised glucose tolerance in INSL5 or RXFP4 knockout mice suggests a metabolic role. This review focuses on the many advances in our understanding of RXFP receptors in the last 5 years, their signal transduction mechanisms, the development of novel compounds that target RXFP1–4, the challenges facing the field, and current prospects for new therapeutics.

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.

Coronary constriction and consequent cardiodepression in pulmonary embolism are mediated by pulmonary big endothelin and enhanced in early endothelial dysfunction.

OBJECTIVES Myocardial ischemia plays a central role in the development of right ventricular failure after acute pulmonary embolism. This study investigates whether pulmonary mediators act specifically on coronary tone and cardiac contractile function in acute pulmonary microembolization and whether such effects are altered in the case of early systemic atherosclerosis. We employ a novel model of serial perfusion in which an isolated rabbit heart is perfused with the effluent of the same animals isolated lung. DESIGN Controlled experiment using isolated organs. SETTING Experimental laboratory. SUBJECTS Male New Zealand White rabbits (controls). Age-matched, male Watanabe rabbits (hypercholesterolemic, development of accelerated atherosclerosis). INTERVENTIONS Seven isolated control and seven isolated Watanabe hearts were perfused with the saline effluent of the same animals isolated lung. After the assessment of the baseline data, the lungs were gradually embolized with glass beads measuring 100 microm in diameter to induce an increase in mean pulmonary arterial pressure from 6 to 8 mm Hg, at baseline, up to 25 mm Hg. MEASUREMENTS AND MAIN RESULTS Pulmonary embolization to 25 mm Hg evoked a coronary constriction, measured as coronary flow decrease to 89 +/- 7% of the baseline value in controls. In the Watanabe group, coronary constriction was significantly enhanced, compared with controls, with coronary flow decreasing to 76 +/- 6% of the baseline value. In both groups, coronary constriction was followed by a deterioration in cardiac contractile performance. This cardiodepression was significantly deeper in Watanabe hearts with respect to both maximum ventricular pressures and maximum rates of pressure development and decline. Coronary constriction and cardiodepression were prevented by coronary infusion of the nonselective endothelin antagonist PD-145065, the endothelinA antagonists A-127722 and BQ-123, and the endothelin-converting enzyme inhibitor phosphoramidon. Concentration of big endothelin in pulmonary effluent increased from 5.6 +/- 0.3 pmol/L in controls and 5.6 +/- 0.2 pmol/L in the Watanabe group, at baseline, to 8.8 +/- 0.4 pmol/L in controls and 8.9 +/- 0.4 pmol/L in the Watanabe group, at 25 mm Hg pulmonary arterial pressure. Endothelin was not detectable at any time during the experiment in pulmonary effluent. The coronary gradient, calculated as a difference in concentration between coronary and pulmonary effluent, was negative for big endothelin and positive for endothelin in both groups. CONCLUSIONS We have demonstrated that an increase in pulmonary release of big endothelin occurs during lung embolism, which, in turn, results in coronary constriction and consequent cardiodepression. This action of big endothelin is based on its local coronary conversion into endothelin. In addition, coronary endothelial dysfunction, attributed to early systemic atherosclerosis, was shown to represent a specific risk factor in these events.