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 (EP1,2,3,4). 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.
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.
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.
These data suggest EP4 receptor agonists as potential therapeutic intervention for diseases with increased vascular permeability such as acute lung injury.