Spatiotemporal EP4-fibulin-1 expression is associated with vascular intimal hyperplasia

Shigekuni Okumura; Sayuki Oka; Takako Sasaki; Marion A Cooley; Yuko Hidaka; Hana Inoue; Hitoshi Nishijima; Shin-Ichiro Ohno; Shota Tanifuji; Mari Kaneko; Takaya Abe; Masahiko Kuroda; Tadashi Yokosuka; Richard M Breyer; Hiroshi Homma; Yuko Kato; Utako Yokoyama

doi:10.1093/cvr/cvae211

Spatiotemporal EP4-fibulin-1 expression is associated with vascular intimal hyperplasia

Cardiovasc Res. 2024 Sep 22:cvae211. doi: 10.1093/cvr/cvae211. Online ahead of print.

Authors

Shigekuni Okumura^{1

2}, Sayuki Oka¹, Takako Sasaki³, Marion A Cooley⁴, Yuko Hidaka¹, Hana Inoue¹, Hitoshi Nishijima⁵, Shin-Ichiro Ohno⁶, Shota Tanifuji¹, Mari Kaneko⁷, Takaya Abe⁷, Masahiko Kuroda⁶, Tadashi Yokosuka⁵, Richard M Breyer⁸, Hiroshi Homma², Yuko Kato^{1

9}, Utako Yokoyama¹

Affiliations

¹ Department of Physiology, Tokyo Medical University, Tokyo, Japan.
² Department of Emergency and Critical Care Medicine, Tokyo Medical University, Tokyo, Japan.
³ Department of Pharmacology, Faculty of Medicine, Oita University, Oita, Japan.
⁴ Department of Oral Biology and Diagnostic Sciences, Augusta University, GA, USA.
⁵ Department of Immunology, Tokyo Medical University, Tokyo, Japan.
⁶ Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan.
⁷ Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan.
⁸ Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA.
⁹ Department of Advanced Medical Science, Faculty of Medicine, Oita University, Oita, Japan.

PMID: 39305517
DOI: 10.1093/cvr/cvae211

Abstract

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 signaling 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 signaling 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 signaling 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.

Conclusions: EP4 was upregulated in VSMCs of proliferative IH, and EP4 signaling promoted IH, at least in part through fibulin-1. An EP4 antagonist might be considered as a therapeutic strategy for IH.

Keywords: fibulin-1; neointima; prostaglandin E; prostanoid receptor EP4; vascular injuries.

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