Abstract 13042: EP4 Signaling in Smooth Muscle Cells Attracts Inflammatory Immune Responses in the Aorta

Abstract

Chronic inflammation driven by prostaglandin E 2 (PGE 2 ) is a basis for vascular diseases such as abdominal aortic aneurysm (AAA). We have reported that PGE 2 receptor, EP4, is responsible for AAA progression. However, precise molecular mechanisms are unknown. Since EP4 expression was upregulated in smooth muscle cells (SMCs) in human AAA, we hypothesized that EP4 in SMCs promotes vascular inflammatory responses in AAA. Methods and Results: EP4 transgenic mice (EP4-Tg) in which EP4 is overexpressed selectively in SMCs under SM22α promoter were generated using Cre-loxP system. Neither EP4-Tg nor littermate non-transgenic mice (Non-Tg) exhibited hypertension, hyperlipidemia, or vascular anomaly at basal condition (male, 12-16 weeks-old). Angiotensin II (AngII) infusion (1.0μg/kg/min) caused AAA rupture-related death in all the EP4-Tg around day14 of infusion, while no AAA were observed in Non-Tg until day28 ( n =6-7, P <0.001). To elucidate initial events leading to AAA, the aortae were analyzed at day4 of AngII infusion, at which no AAA was observed. In EP4-Tg aortae, MMP-9 activity was induced ( n =6-7, P <0.05) and IL-6 production was upregulated (5.0±1.8-fold vs. Non-Tg, n =6-7, P <0.05). FACS analysis showed that infiltration of Ly6C hi inflammatory monocytes was enhanced in EP4-Tg aortae, coinciding with the emergence of MMP-9 expression in resident macrophages but not in Non-Tg counterpart (n=4, P <0.01). IL-6 expression was detected primarily in SMC layer in EP4-Tg by immunohistochemistry. Administration of IL-6R antibody (MR16-1, 10mg/kg/2days) in vivo inhibited AngII-induced infiltration of Ly6C hi monocytes (0.3±0.1-fold, n =4, P <0.05) and AAA rupture in EP4-Tg ( n =6, P =0.06). Administration of EP4 antagonist (ONO AE3-208, 0.1mg/kg, BID) also inhibited AngII-induced AAA in EP4-Tg ( n =8, P =0.05). In vitro , EP4 stimulation induced more abundant IL-6 production in EP4-Tg aortic SMCs than Non-Tg SMCs (33.0±4.8-fold, n =4, P <0.001), which was attenuated by PKA inhibitor or TAK1 inhibitor. Immunohistochemistry showed abundant expressions of IL-6 and phosphorylated TAK1 in EP4-positive human AAA but not in non-aneurysmal aorta. Conclusion: PGE 2 -EP4 signaling in SMCs initiates inflammatory reaction and AAA formation via upregulation of IL-6.