Mechanisms of allergy and clinical immunology
Endothelial E-type prostanoid 4 receptors promote barrier function and inhibit neutrophil trafficking

https://doi.org/10.1016/j.jaci.2012.05.008Get rights and content

Background

Increased vascular permeability is a fundamental characteristic of inflammation. Substances that are released during inflammation, such as prostaglandin (PG) E2, can counteract vascular leakage, thereby hampering tissue damage.

Objective

In this study we investigated the role of PGE2 and its receptors in the barrier function of human pulmonary microvascular endothelial cells and in neutrophil trafficking.

Methods

Endothelial barrier function was determined based on electrical impedance measurements. Neutrophil recruitment was assessed based on adhesion and transendothelial migration. Morphologic alterations are shown by using immunofluorescence microscopy.

Results

We observed that activation of E-type prostanoid (EP) 4 receptor by PGE2 or an EP4-selective agonist (ONO AE1-329) enhanced the barrier function of human microvascular lung endothelial cells. EP4 receptor activation prompted similar responses in pulmonary artery and coronary artery endothelial cells. These effects were reversed by an EP4 antagonist (ONO AE3-208), as well as by blocking actin polymerization with cytochalasin B. The EP4 receptor–induced increase in barrier function was independent of the classical cyclic AMP/protein kinase A signaling machinery, endothelial nitric oxide synthase, and Rac1. Most importantly, EP4 receptor stimulation showed potent anti-inflammatory activities by (1) facilitating wound healing of pulmonary microvascular endothelial monolayers, (2) preventing junctional and cytoskeletal reorganization of activated endothelial cells, and (3) impairing neutrophil adhesion to endothelial cells and transendothelial migration. The latter effects could be partially attributed to reduced E-selectin expression after EP4 receptor stimulation.

Conclusion

These data indicate that EP4 agonists as anti-inflammatory agents represent a potential therapy for diseases with increased vascular permeability and neutrophil extravasation.

Section snippets

Culture of endothelial cells

Human lung microvascular endothelial cells (HMVEC-Ls), human pulmonary artery endothelial cells (HPAECs), and human coronary artery endothelial cells (HCAECs) were purchased from Lonza (Verviers, Belgium). HMVEC-Ls and HCAECs were maintained in EGM-2 MV BulletKit medium (Lonza) with 5% FCS. HPAECs were maintained in EGM-2 BulletKit medium with 2.5% FCS. Endothelial cells were cultivated as previously described.12, 13 For further details on reagents, see the Methods section in this article's

Stimulation of EP4 receptors enhances endothelial barrier function

The selective EP4 agonist ONO AE1-329 (ONO Pharmaceutical, Osaka, Japan) and PGE2 concentration-dependently (10-300 nmol/L) increased endothelial barrier function, which was recorded as electrical impedance, in HMVEC-Ls and, to a lesser extent, in macrovascular HPAECs (Fig 1, A and B). In contrast, the barrier-enhancing effect of S1P (Fig 1, C) was less pronounced in HMVEC-Ls compared with that seen in HPAECs. Specific involvement of the EP4 receptor was demonstrated by preincubating the

Discussion

Our results demonstrate that activation of the PGE2 receptor EP4 increases basal barrier function and prevents barrier dysfunction induced by edemagenic stimuli. We found that the EP4-selective agonist ONO AE1-329 and PGE2 itself concentration-dependently increased the barrier function of human pulmonary microvascular and macrovascular endothelial cells, as indicated by an increase in electrical impedance. The effects of both PGE2 and the EP4 agonist were completely prevented by the selective

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    V.K., A.Ü., and S.P. were funded by the PhD Program Molecular Medicine of the Medical University of Graz. This work was supported by the Start Funding Program of the Medical University of Graz (ASO109000101 to V.K.), the Austrian Science Fund FWF (grants P22521 to A.H., P21004 to G.M., and SFB Lipotox project 3007 to W.S.), and Jubiläumsfonds of the Austrian National Bank (14263 to A.H. and 13487 to R.S.).

    Disclosure of potential conflict of interest: A. Heinemann has received one or more payments for lecturing from AstraZeneca AB (Sweden), is a Principal Investigator for the Austria Science Funds (FWF) and for Jubilaümfonds of the Austrian National Bank (OeNB), and has received research funding from AstraZeneca AB (Sweden) and from Alimrall SA (Spain). The rest of the authors declare that they have no relevant conflicts of interest.

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