Multiple roles of the PGE2 -EP receptor signal in vascular permeability

Abstract

Background and purpose: PGE2 is a major prostanoid that regulates inflammation by stimulating EP1-4 receptors. However, how PGE2 induces an initial inflammatory response to vascular hyper-permeability remains unknown. Here we investigated the role of the PGE2 -EP receptor signal in modulating vascular permeability both in vivo and in vitro.

Experimental approach: We used a modified Miles assay and intravital microscopy to examine vascular permeability in vivo. Endothelial barrier property was assessed by measuring transendothelial electrical resistance (TER) in vitro.

Key results: Local administration of PGE2 , an EP2 or EP4 receptor agonist into FVB/NJcl mouse ear skin caused vascular leakage, indicated by dye extravasation. Intravital microscopy and laser Doppler blood-flow imaging revealed that these treatments dilated peripheral vessels and increased local blood flow. Pretreatment with the vasoconstrictor phenylephrine inhibited the PGE2 -induced blood flow increase and vascular leakage. In contrast to the EP2 and EP4 receptor agonists, administration of an EP3 receptor agonist suppressed vascular leakage without altering vascular diameter or blood flow. In isolated HUVECs, the EP3 receptor agonist elevated TER and blocked thrombin-induced dextran passage. Inhibiting PKA restored the hypo-permeability induced by the EP3 receptor agonist.

Conclusions and implications: Activation of the PGE2 -EP2 or -EP4 receptor signal induces vasodilatation in mural cells, resulting in increased local blood flow and hyper-permeability. In contrast, activation of the PGE2 -EP3 receptor signal induces a cAMP-dependent enhancement of the endothelial barrier, leading to hypo-permeability. We provide the first evidence that endothelial cells and mural cells cooperate to modulate vascular permeability.

Figure 1

Effect of PGE₂ or EP receptor agonists on vascular permeability in vivo. (A) Representative pictures of Evans Blue extravasation. Treatment with vehicle (left ear), PGE₂ (right ear), EP₁ receptor agonist (ONO-DI-004), EP₂ receptor agonist (ONO-AE1-259-01), EP₃ receptor agonist (ONO-AE-248) or EP₄ receptor agonist (ONO-AE1-329). (B) Effect of PGE₂ and EP receptor agonists on Evans Blue dye extravasation (n = 6). (C) Representative pictures of VEGF-induced Evans Blue extravasation. Treatment with VEGF (upper panel, left ear), VEGF and PGE₂ (upper panel, right ear), or VEGF and each EP receptor agonist (middle and lower panels). (D) Effect of PGE₂ and EP receptor agonists on VEGF-induced Evans Blue dye extravasation (n = 6). (E) Representative pictures of PGE₂-induced Evans Blue extravasation. Treatment with PGE₂ (left ear), PGE₂ and EP₂ receptor antagonist (PF 04418948, right ear), PGE₂ and EP₃ receptor antagonist (L798106, right ear), or PGE₂ and EP₄ receptor antagonist (AH23848, right ear). (F) Effect of EP_2-4 receptor antagonists on PGE₂-induced Evans Blue extravasation (4 ≤ n ≤ 12). * P < 0.05, ** P < 0.01significantly different from the results in vehicle treatment. Data are presented as means ± SEM.

Figure 2

PGE₂, EP₂ and EP₄ receptor agonists increase local blood flow in vivo. (A) Illustration of mouse left ear vessel. (B) Typical pictures of whole mount immunostaining of PECAM (left panels, green), αSMA (upper middle panel, red), and desmin (lower middle panel, red). Right panels show merged pictures of PECAM, αSMA or desmin, and DAPI (blue) staining. Scale bar, 100 μm. (C) Typical pictures of changes in local blood flow. Treatment with vehicle (upper panel, left ear), PGE₂ (upper panel, right ear), and EP_2-4 receptor agonists (lower panels). (D) Effect of PGE₂ or EP_2-4 receptor agonists on blood flow change (n = 5). * P < 0.05, ** P < 0.01 significantly different from the results of the vehicle treatment. Data are presented as means ± SEM.

Figure 3

PGE₂, EP₂ and EP₄ receptor agonist increase vascular diameter in vivo. (A) Representative pictures of PGE₂-induced vascular diameter increase. Vascular diameter was measured as indicated by the red double-headed arrows. A, indicates artery and V indicates vein. Scale bar, 50 μm. (B) Effect of PGE₂ and EP_2-4 receptor agonists on vascular diameter of proximal vessels (n = 4). (C) Effect of PGE₂ and EP_2-4 receptor agonists on vascular diameter of distal vessels (n = 4). (D) Content of cAMP in sections of endothelium-denuded mouse aorta. The sections were treated with PGE₂ or EP_2-4 receptor agonists (n = 4). *P < 0.05, **P < 0.01 significantly different from the results of the vehicle treatment. Data are presented as means ± SEM.

Figure 4

Phenylephrine suppresses PGE₂-induced vasodilatation and hyper-permeability. (A) Representative pictures of Evans Blue dye extravasation. Treatment with PGE₂ with (right ear) or without (left ear) phenylephrine (Phe) pretreatment. (B) Representative pictures of changes in local blood flow. (C) Effect of Phe on PGE₂-induced vasodilatation in proximal vessels (n = 4). (D) Effect of Phe on PGE₂-induced vasodilatation in distal vessels (n = 4). *P < 0.05, **P < 0.01 significantly different from the results of the vehicle treatment. ^#P < 0.05, ^##P < 0.01 significantly different from the results of PGE₂ stimulation with vehicle pretreatment (C, D). Data are presented as means ± SEM.

Figure 5

Effect of PGE₂ and EP receptor agonists on endothelial barrier function in vitro. (A) Effect of PGE₂ on TER. (B) Maximum TER increase induced by PGE₂ or EP receptor agonist (6 ≤ n ≤ 12). (C) Maximum TER increase induced by PGE₂ under EP blockade (n = 6). *P < 0.05, **P < 0.01 significantly different from the results of the vehicle treatment. ^#P < 0.05, ^##P < 0.01 significantly different from the results in PGE₂ treatment. Data are presented as means ± SEM.

Figure 6

EP₃ receptor agonism enhances the endothelial barrier in vitro. (A) HUVECs were transfected with either control or EP₃ receptor siRNA. The maximum increase in TER induced by EP₃ receptor agonist, PGE₂, or sphingosine-1-phosphate (S1P) was quantified (6 ≤ n ≤ 9). (B) FITC-dextran permeability assay (5 ≤ n ≤ 6). (C) Typical pictures of immunostaining of VE-cadherin (left panels, green) and F-actin (middle panels, red) after thrombin stimulation with and without EP₃ receptor agonist. Right panels show merged pictures of VE-cadherin, F-actin, and DAPI (blue) staining. Scale bar is 10 μm. (D) Measurement of intracellular cAMP level in HUVECs after stimulation of EP₃ receptors (n = 4). (E) Effects of PKAi on EP₃ receptor-induced increases in TER (n = 6). **P < 0.01 significantly different from the results of the vehicle treatment. ^#P < 0.05, ^##P < 0.01 significantly different from the results in HUVECs infected with control siRNA (A) or after stimulation by EP₃ receptor agonist without any pretreatment (B, D). Data are presented as means ± SEM.