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

Abstract Aims Cyclooxygenase-2– and microsomal prostaglandin E synthase-1–derived prostaglandin E 2 (PGE 2 ) are involved in vascular intimal hyperplasia (IH). Although extensive studies have revealed the roles of PGE 2 receptors (EPs) in IH, spatiotemporal EP expressions and downstream targets have not been fully elucidated. In this study, we focused on EP4 and investigated its role in vascular IH. Methods and Results We generated EP4 reporter mice ( Ptger4 -IRES-nlsLacZ) and found prominent EP4 expression in the proliferative neointima 2 weeks after femoral artery wire injury. Expression of EP4 were returned to the baseline level 4 weeks after vascular injury (VI). Injury-induced IH was diminished in vascular smooth muscle cell (VSMC)-specific EP4 heterozygous deficient mice ( Ptger4 fl/+ ; SM22 - Cre ) 2 and 4 weeks after VI compared to SM22 - Cre , whereas injury-induced IH was exacerbated in VSMC-specific EP4-overexpressing mice ( Ptger4 -Tg) compared to controls (non-Tg). Systemic EP4 antagonist administration reduced VI-induced IH in wild-type mice. We investigated the role of extracellular matrix proteins, as downstream regulated targets of EP4. Stimulation of EP4 increased mRNA and protein levels of fibulin-1 (a multifunctional glycoprotein) in Ptger4 -Tg VSMCs. Fibulin-1C or -1D recombinant proteins increased VSMC proliferation, whereas proliferation was decreased in fibulin-1–deficient 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 upregulated by EP4 stimulation, and fibulin-1 and ECM1 proteins additively enhanced VSMC proliferation. Similar to EP4 expression, both fibulin-1 and ECM1 were abundantly expressed in the neointima 2 weeks after VI. Furthermore, injury-induced IH was attenuated in VSMC-specific fibulin-1 deletion mice ( Fbln1 fl/fl ; SM22 -Cre) compared to Fbln1 fl/fl . Conclusions EP4 was upregulated in proliferative IH, and EP4-induced fibulin-1 cooperated with ECM1 to promote IH through VSMC proliferation. The calcium binding EGF-like modules 6-8 of fibulin-1 are indicated to regulate cell proliferation. A Translational Perspective Recent advances in drug-eluting stents have significantly contributed to the reduction of vascular IH. However, the detailed mechanism underlying IH after stenting remains to be elucidated. We found that prostaglandin E 2 -EP4–induced fibulin-1 plays a role in IH through VSMC proliferation. It is well recognized that prostaglandin E 2 plays a role in IH, but inhibition of cyclooxygenase-2 has side effects such as thrombogenesis. Because EP4 and fibulin-1 were upregulated specifically in the neointima after vascular injury, oral or local administration of an EP4 antagonist or the downregulation of fibulin-1 would be potential therapeutic strategies to restrain IH.