The role of PGE₂ EP₄receptors in the regulation of endothelial barrier function

Background

Prostaglandin E₂ plays a crucial role in inflammation, including pain, fever and tumorigenesis. Inflammatory cells, fibroblasts and epithelium are the main source of PGE₂ throughout an immune response. There are four known receptors for PGE₂, E type prostanoid receptors 1–4 (EP_1,2,3,4). According to recent reports, the EP₄ receptor seems to be a potential target of therapeutic treatment for attenuating inflammation as well as increased endothelial permeability.

Methods

Primary human microvascular endothelial cells of the lung (HMVEC-L) were cultured and transfected using siRNA approach. The mRNA level of the EP₄ receptor was determined using RT-PCR. EP₄ receptor protein expression was evaluated via Western blotting and flow cytometry. Changes in electrical impedance were measured using an ECIS application. Morphological alterations were observed via immunofluorescent staining of β-catenin and F-actin. Cell cycle and apoptosis analysis were performed using flow cytometry.

Results

The pulmonary microvascular endothelial cells express the PGE₂ receptor EP₄, which was shown by flow cytometry and Western blotting. In endothelial cells, EP₄ receptor protein expression was down-regulated to less than 40% by using the siRNA transfection approach. Also, the mRNA level of the EP₄ receptor was significantly down-regulated below 15% using siRNA transfection. In the endothelial impedance measurements, the EP₄ agonist and PGE₂-induced barrier enhancement was significantly suppressed in EP₄ receptor-silenced cells. Morphological studies revealed that the thrombin-induced disruption of endothelial monolayers could be reversed by stimulation of EP₄ receptors. Cell-cell contacts were enhanced and stress fibre formation was prevented by pre-treatment with PGE₂ and an EP₄-selective agonist. PGE₂ and the EP₄ agonist did not induce any changes in the endothelial cell cycle; however, EP₄ receptor activation appeared to be protective against staurosporine-induced apoptosis. Apoptotic cells were determined in the sub-G₁ phase of the cell cycle.

Conclusions

These data suggest EP₄ receptor agonists as potential therapeutic intervention for diseases with increased vascular permeability such as acute lung injury.