Aims Cyclooxygenase-2–derived prostaglandin E2 (PGE2) is thought to promote vascular intimal hyperplasia (IH). It has been reported that the PGE2 receptor EP4 is upregulated in injured vessels and that EP4 signalling in vascular smooth muscle cells (VSMCs) promotes IH. In contrast, EP4 in endothelial cells has been demonstrated to restrain IH. We aimed to investigate spatiotemporal expression of EP4 and whether modulating EP4 signalling could be a viable therapeutic strategy. Methods and results We generated EP4 reporter mice (Ptger4-IRES-nlsLacZ) and found temporary but prominent EP4 expression in VSMCs of the proliferative neointima 2 weeks after femoral artery wire injury. Injury-induced IH was diminished in VSMC-targeted EP4 heterozygous deficient mice (Ptger4fl/+;SM22-Cre) 2 and 4 weeks after vascular injury compared to that in SM22-Cre, whereas injury-induced IH was exacerbated in VSMC-targeted EP4-overexpressing mice (Ptger4-Tg) compared to controls (non-Tg). We then investigated the downstream signalling of EP4 in VSMCs. Stimulation of EP4 increased mRNA and protein levels of the glycoprotein fibulin-1 in Ptger4-Tg VSMCs. Fibulin-1C recombinant proteins increased VSMC proliferation and migration through transforming growth factor (TGF)- β/Smad3, and EP4-mediated proliferation and migration were attenuated in Fbln1fl/fl;SM22-Cre VSMCs and in CRISPR/Cas9-mediated Fbln1 knockdown in Ptger4-Tg VSMCs. We generated multiple deletion mutants of fibulin-1C and found that EGF-like modules 6–8 appear to be involved in fibulin-1-mediated proliferation. Among binding partners of fibulin-1, extracellular matrix protein 1 (ECM1) was also upregulated by EP4 stimulation, and fibulin-1C and ECM1 proteins additively enhanced VSMC proliferation and migration. Injury-induced IH was attenuated in VSMC-targeted fibulin-1 deletion mice (Fbln1fl/fl;SM22-Cre) compared to Fbln1fl/fl. Furthermore, systemic EP4 antagonist administration reduced injury-induced IH in wild-type mice. Conclusion EP4 was upregulated in VSMCs of proliferative IH, and EP4 signalling promoted IH, at least in part through fibulin-1. An EP4 antagonist might be considered as a therapeutic strategy for IH.

中文翻译：

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时空 EP4-fibulin-1 表达与血管内膜增生有关


目的 环氧合酶-2 衍生的前列腺素 E2 （PGE2） 被认为可促进血管内膜增生 （IH）。据报道，PGE2 受体 EP4 在受伤血管中上调，血管平滑肌细胞 （VSMC） 中的 EP4 信号传导促进 IH。相比之下，内皮细胞中的 EP4 已被证明可以抑制 IH。我们旨在研究 EP4 的时空表达以及调节 EP4 信号传导是否是一种可行的治疗策略。方法和结果：我们生成了 EP4 报告小鼠 （Ptger4-IRES-nlsLacZ），发现股动脉导线损伤后 2 周在增殖性新内膜的 VSMC 中临时但显着的 EP4 表达。在 VSMC 靶向 EP4 杂合缺陷小鼠 （Ptger4fl/+;SM22-Cre） 与血管损伤相比，血管损伤后 2 周和 4 周，而与对照 （非 Tg） 相比，VSMC 靶向 EP4 过表达小鼠 （Ptger4-Tg） 损伤诱导的 IH 加剧。然后，我们研究了 EP4 在 VSMC 中的下游信号传导。刺激 EP4 增加了 Ptger4-Tg VSMCs 中糖蛋白 fibulin-1 的 mRNA 和蛋白水平。Fibulin-1C 重组蛋白通过转化生长因子 （TGF）-β/Smad3 增加了 VSMC 增殖和迁移，EP4 介导的增殖和迁移在 Fbln1fl/fl 中减弱;SM22-Cre VSMC 和 Ptger4-Tg VSMC 中 CRISPR/Cas9 介导的 Fbln1 敲低。我们生成了 fibulin-1C 的多个缺失突变体，发现 EGF 样模块 6-8 似乎参与 fibulin-1 介导的增殖。在 fibulin-1 的结合伴侣中，细胞外基质蛋白 1 （ECM1） 也因 EP4 刺激而上调，fibulin-1C 和 ECM1 蛋白相加增强 VSMC 增殖和迁移。 在 VSMC 靶向 fibulin-1 缺失小鼠 （Fbln1fl/fl;SM22-Cre） 与 Fbln1fl/fl 的比较。此外，全身性 EP4 拮抗剂给药减少了野生型小鼠损伤诱导的 IH。结论 EP4 在增殖性 IH 的 VSMC 中上调，EP4 信号传导至少部分通过 fibulin-1 促进 IH。EP4 拮抗剂可能被视为 IH 的治疗策略。