Relaxin inhibits early steps in vascular inflammation Anna Brecht ⁎, Cornelia Bartsch, Gert Baumann, Karl Stangl, Thomas Dschietzig Department of Cardiology and Angiology, Charité – University Medicine Berlin, Campus Mitte, Schumannstr. 20/21, 10117 Berlin, Germany abstract article info Article history: Received 3 May 2010 Received in revised form 22 August 2010 Accepted 13 September 2010 Available online 17 September 2010 Keywords: Relaxin TNF-α Vascular inflammation Endothelial adhesion molecules MCP-1 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 ath- erosclerosis and of other pro-inflammatory states. © 2010 Elsevier B.V. All rights reserved. 1. Introduction The relaxin hormone belongs to the relaxin peptide family, encoded by the insulin-like peptide genes and by the relaxin genes [1]. The relaxin-2 peptide, the major circulating relaxin subtype in humans, is composed of two chains with two interchain and one intrachain disulphide bonds [2]. In 2002, specific relaxin receptors were found by Hsu et al., the relaxin family peptide receptor1 (RXFP1) and the RXFP2 [3]. RXFP1 signalling is complex involving multiple pathways depending on the cell type under investigation, but at least two major cascades are abundant: the cyclic AMP (cAMP) and the nitric oxide pathways [3–5]. In the last years, however, there has been evidence that relaxin exerts, next to RXFP1 signalling, also hemody- namic and anti-inflammatory effects as a functional endothelin-1 antagonist and as an agonist at the glucocorticoid receptor (GR) [6,7]. Even though exact mechanisms regarding relaxin signalling pathways remain debatable, there is clear evidence assigning relaxin a cardio- protective role due to its anti-fibrotic, vasodilatory, and anti-ischemic characteristics [1,8–12]. Intimal thickening, deposition of atherogenic lipoproteins, and inflammatory activation form early steps in the development of atherosclerosis, resulting in endothelial activation by upregulation of specific adhesion molecules like VCAM-1, ICAM-1 and E-selectin. This leads to monocyte rolling and tethering to the vascular endothe- lium and increased expression of PECAM, which triggers leukocyte diapedesis and infiltration of inflammatory cells to the subendothelial layer. Cytokines and chemokines, like TNF-α and the monocyte chemo- attractant protein-1 (MCP-1), are central mediators in initiating and sustaining monocyte chemotaxis, adhesion and diapedesis and might therefore represent central therapeutic targets in vascular inflamma- tory states, such as early atherosclerosis [13], vasculitis [14], and other states of vascular inflammation such as pulmonary airway inflamma- tion [15] and glomerulonephritis [16]. The adhesion molecule VCAM-1 is expressed on activated vascular endothelium and mediates, through specific binding to the monocyte receptor VLA-4, firm monocyte adhesion and infiltration into the intima, where foam cells are formed by phagocytosis of lipoprotein remnants [17]. There is increasing evidence that relaxin, next to its tissue- modulating and hemodynamic effects, fulfils also immunomodulatory effects, such as suppressing endotoxin-stimulated cytokine expres- sion of THP-1 cells, mediated through binding and activation of the GR [7]. In this study, we provide evidence that relaxin mitigates early vascular inflammatory events, such as upregulation of the adhesion Regulatory Peptides 166 (2011) 76–82 ⁎ Corresponding author. Tel.: +49 30 450 613 006; fax: +49 30 450 513 916. E-mail addresses: anna.brecht@charite.de (A. Brecht), cornelia.bartsch@charite.de (C. Bartsch), gert.baumann@charite.de (G. Baumann), karl.stangl@charite.de (K. Stangl), thomas.dschietzig@charite.de (T. Dschietzig). 0167-0115/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2010.09.001 Contents lists available at ScienceDirect Regulatory Peptides journal homepage: www.elsevier.com/locate/regpep