Okumura Shigekuni, Oka Sayuki, Sasaki Takako, Cooley Marion A, Hidaka Yuko, Inoue Hana, Nishijima Hitoshi, Ohno Shin-Ichiro, Tanifuji Shota, Kaneko Mari, Abe Takaya, Kuroda Masahiko, Yokosuka Tadashi, Breyer Richard M, Homma Hiroshi, Kato Yuko, Yokoyama Utako
Department of Physiology, Tokyo Medical University, Tokyo, Japan.
Department of Emergency and Critical Care Medicine, Tokyo Medical University, Tokyo, Japan.
Cardiovasc Res. 2024 Dec 31;120(17):2293-2306. doi: 10.1093/cvr/cvae211.
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.
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.
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.
环氧化酶-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在受损血管中上调，并且血管平滑肌细胞（VSMC）中的EP4信号传导促进IH。相反，已证明内皮细胞中的EP4可抑制IH。我们旨在研究EP4的时空表达以及调节EP4信号传导是否可能是一种可行的治疗策略。
我们构建了EP4报告基因小鼠（Ptger4-IRES-nlsLacZ），并发现股动脉钢丝损伤后2周，增殖性新生内膜的VSMC中存在短暂但显著的EP4表达。与SM22-Cre小鼠相比，血管损伤后2周和4周，VSMC靶向的EP4杂合缺陷小鼠（Ptger4fl/+;SM22-Cre）中损伤诱导的IH减少，而与对照（非Tg）相比，VSMC靶向的EP4过表达小鼠（Ptger4-Tg）中损伤诱导的IH加剧。然后，我们研究了VSMC中EP4的下游信号传导。刺激EP4可增加Ptger4-Tg VSMC中糖蛋白纤连蛋白-1的mRNA和蛋白质水平。纤连蛋白-1C重组蛋白通过转化生长因子（TGF）-β/Smad3增加VSMC增殖和迁移，并且在Fbln1fl/fl;SM22-Cre VSMC以及Ptger4-Tg VSMC中CRISPR/Cas9介导的Fbln1敲低中，EP4介导的增殖和迁移减弱。我们构建了纤连蛋白-1C的多个缺失突变体，发现表皮生长因子样模块6-8似乎参与纤连蛋白-1介导的增殖。在纤连蛋白-1的结合伴侣中，细胞外基质蛋白1（ECM1）也被EP4刺激上调，并且纤连蛋白-1C和ECM1蛋白可累加增强VSMC增殖和迁移。与Fbln1fl/fl相比，VSMC靶向的纤连蛋白-1缺失小鼠（Fbln1fl/fl;SM22-Cre）中损伤诱导的IH减弱。此外，全身性给予EP4拮抗剂可减少野生型小鼠中损伤诱导的IH。
增殖性IH的VSMC中EP4上调，并且EP4信号传导促进IH，至少部分是通过纤连蛋白-1。EP4拮抗剂可能被视为IH的一种治疗策略。
