Identification of chemerin receptor (ChemR23) in human endothelial cells: Chemerin-induced endothelial angiogenesis

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Abstract

Chemerin acting via its distinct G protein-coupled receptor CMKLR1 (ChemR23), is a novel adipokine, circulating levels of which are raised in inflammatory states. Chemerin shows strong correlation with various facets of the metabolic syndrome; these states are associated with an increased incidence of cardiovascular disease (CVD) and dysregulated angiogenesis. We therefore, investigated the regulation of ChemR23 by pro-inflammatory cytokines and assessed the angiogenic potential of chemerin in human endothelial cells (EC). We have demonstrated the novel presence of ChemR23 in human ECs and its significant up-regulation (P < 0.001) by pro-inflammatory cytokines, TNF-α, IL-1β and IL-6. More importantly, chemerin was potently angiogenic, as assessed by conducting functional in-vitro angiogenic assays; chemerin also dose-dependently induced gelatinolytic (MMP-2 & MMP-9) activity of ECs (P < 0.001). Furthermore, chemerin dose-dependently activated PI3K/Akt and MAPKs pathways (P < 0.01), key angiogenic and cell survival cascades. Our data provide the first evidence of chemerin-induced endothelial angiogenesis and MMP production and activity.

Introduction

Dysregulated angiogenesis is the hallmark of cardiovascular diseases (CVD), with obesity and metabolic syndrome (MS) being significant contributors to CVD [1]. The metabolic syndrome is associated with excessive accumulation of central body fat. Adipose tissue produces several hormones and cytokines termed ‘adipokines’ having widespread metabolic effects on vascular endothelium [2], [3]. Adipokines also appear to play important roles in the pathogenesis of insulin resistance, diabetes, and atherosclerosis [4]. Moreover, modulation of neo-angiogeneic responses of adipokines has been convincingly demonstrated within adipose tissue, further establishing the link between MS and CVD [5].

Chemerin is a recently discovered 16-kDa adipokine and chemoattractant protein that serves as a ligand for the G protein-coupled receptor, CMKLR1 (ChemR23), with a role in adaptive and innate immunity [6], [7]. Furthermore, chemerin is elevated in obesity and shows strong correlation with various facets of the MS, including dyslipidaemia and hypertension; we have recently shown serum and adipose tissue chemerin levels to be increased in women with MS [8], [9]. Others, have reported elevated levels of circulating chemerin in inflammatory states, such as subjects with rheumatoid arthritis who are reported to have increased CVD; inflammation being a key player in immune mediated atherosclerosis [10], [11].

Recently, the chemerin/ChemR23 system has been implicated in mediating cellular migration under inflammatory conditions [12], a prerequisite of endothelial angiogenesis. This is of interest, as it is increasingly evident from the literature that adipokines play a significant role in the induction of atherogenesis and dysregulated angiogenesis [13], [14]. However, no studies to date have described the presence of ChemR23 in human endothelial cells (ECs) and its role in endothelial angiogenesis.

With the aforementioned, we sought to investigate the possible interplay between chemerin/ChemR23 system and the human endothelium. In the present study, we found and report for the first time the presence of ChemR23 in human ECs, and its regulation by pro-inflammatory cytokines. More importantly, chemerin-induced endothelial angiogenesis and induced multiple signalling cascades including MAPK and Akt pathways and activates endothelial gelatinases (MMP-2/9).

Section snippets

Materials and methods

Cell culture and treatments. Human Microvascular Endothelial Cells (HMECs) were obtained from the Centre for Disease Control (CDC) in Atlanta, Georgia, USA. Briefly HMECs were cultured in MCDB medium (Sigma–Aldrich, Dorset, UK) as described previously [15].

For ChemR23 protein expression studies, optimization experiments were carried out by treating serum-starved ECs with or without human recombinant TNF-α (0–20 ng/mL) (NBS biologicals, Cambridgeshire, UK), IL-1β (0–100 ng/mL) (ABCAM,

ChemR23 expression in human endothelium

RT-PCR analysis revealed the presence of ChemR23 mRNA in both micro (HMECs) and macro-vascular human ECs (HUVECs and EA.hy926) (Fig. 1A). Western blotting, using specific chemerin receptor antibody, confirmed its expression in both these cell types as a 42 kDa band (predicted molecular weight) (Fig. 1B). The specificity of which was further confirmed by employing a ChemR23 blocking peptide (data not shown). Additionally, immunocytochemical analysis established the presence and distribution of

Discussion

We describe novel findings, of the presence and regulation of endothelial ChemR23 by pro-inflammatory cytokines. More importantly, we report that chemerin, whose circulating concentrations are altered in obesity and obesity-related disorders, activates key survival and angiogenic signalling cascades like MAPK and Akt pathways. Additionally, we demonstrate for the first time chemerin induced functional angiogenesis in human ECs, by promoting migration and capillary tube formation; and activation

Funding sources

The General Charities of the City of Coventry. Medical Research Fund, Universiy of Warwick.

Disclosures

Authors have no conflict of interest.

Acknowledgment

H.S.R. acknowledges S. Waheguru, University of Warwick, for his continual support.

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    These authors contributed equally to this work.

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