Elsevier

Regulatory Peptides

Volume 158, Issues 1–3, 27 November 2009, Pages 1-5
Regulatory Peptides

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Endothelin receptor A expression in human inflammatory cells

https://doi.org/10.1016/j.regpep.2009.06.004Get rights and content

Abstract

Most inflammatory diseases show elevated levels of endothelin-1 (ET-1) probably due to an alteration in vascular structure and function with activation/accumulation of inflammatory cells. The ET receptors (ETA, ETB) are widely expressed in all human vessels, consistent with the main role of ET-1 in maintaining physiological vascular tone. Previous findings have shown the expression on inflammatory cells such as neutrophils (PMNs) and macrophages (MØs) of ET-1 and endothelin-converting enzyme-1 (ECE-1) (the key enzyme in the biosynthesis of ET-1). Therefore the role of ET-1 cannot be related only to the vasoactivity.

Our study was aimed to determine the expression and the cellular location of ET receptors in both human PMNs and MØs by the use of RT-PCR assay, Western blot analysis and immunocytological methods. Our results showed for the first time that PMNs and MØs clearly expressed ETA (mRNA and protein).

Considering that the overproduction of ET-1 following endothelial dysfunction and inflammation, contributes to pathophysiological processes such as vascular hypertrophy, cell proliferation and fibrosis, our results suggest that PMNs and MØs can also play a key role in vascular dysfunctions via the possible formation of an autocrine loop between ET-1 and ETA.

Introduction

The family of endothelins (ETs), a group of polyfunctional cytokines, is composed of three isoforms (ET-1, ET-2, ET-3) of which the more studied is ET-1, the most potent vasoconstrictor that has also a growth-promoting function [1].

ET-1 is formed by the catalytic action of ECE-1 on precursor big ET-1, and its cellular actions are mediated via ET receptors (ETA, ETB) that are G protein-coupled seven transmembrane-spanning receptors and it is well known that these kinds of receptors are also the major targets for many of the currently available drugs, including cardiovascular and immunomodulatory agents [2]. ETA and ETB receptors are widely expressed in all human vessels, consistent with the main role of ET-1 in maintaining the physiological vascular tone [1].

Furthermore, the peptide is also involved in the pathogenesis of vascular, inflammatory and fibrotic diseases. In particular, many pathological conditions that share an inflammatory component, such as atherosclerosis, pulmonary hypertension [1], [3], sepsis [4], lung diseases [5] and carcinomas [6], show elevated ET-1 levels, probably due to an alteration of the vascular structure with activation/accumulation of inflammatory cells.

The last findings from our group showed the expression of ET-1 and ECE-1 on isolated human PMNs [7] and on PMNs, MØs and mast cells in human inflammated tissues [8], [9], implying a much broader role for this peptide than just vasoactivity. ET-1 participates in the starting and development of inflammatory reaction. Moreover, as a chemotactic factor for MØs [10], it induces leukocyte rolling and adherence to endothelial cells, by enhancing the expression of adhesion molecules through a predominantly ETA receptor-mediated mechanism [11]. Finally, the peptide stimulates the production of inflammatory mediators, like cytokines and growth factors such as the vascular endothelial growth factor (VEGF) [12].

Therefore, the present work was aimed to evaluate for the first time the expression of ET-1 receptors in freshly isolated PMNs and MØs using a more direct approach (by immunofluorescence techniques) than the one present in the literature where up to know the presence of ET receptors in PMNs and MØs has been detected only in an indirect manner. Our data confirm the hypothesis that inflammatory cells present ETA receptor and that, consequently, can be directly activated by ET-1.

Section snippets

Cell culture

The U373, human astrocytoma cell line, was grown in the DMEM (Dulbecco's Modified Eagle's Medium) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal calf serum (FCS) (Sigma), 2 mM l-glutamine, penicillin–streptomycin (10,000 U/ml) and incubated in a humidified 5% CO2 atmosphere at 37 °C in 25 cm2 flasks.

Isolation and treatment of PMNs and monocytes

Human PMNs and lymphomonocytes (PBMCs) were separated from heparinized venous human peripheral blood and were obtained by density gradient Ficoll-Hypaque (Amersham Pharmacia

Results and discussion

In our previous studies we have demonstrated the wide distribution of ET-1 [8] and ECE-1 [9] in inflamed tissues and that this positivity was due to the presence of inflammatory cells such as PMNs, PBMCs and MØs in the tissue.

Furthermore, our group was also able to detect the presence of prepro-ET-1 mRNA in isolated PMNs and to quantify the release of the mature peptide after appropriate stimulation [7].

Thus, as a continuation of our work, this current study evaluates the presence of ET

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1

These authors contributed equally to the study.

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