Spatiotemporal EP4–Fibulin-1 Expression is Associated with Vascular Intimal Hyperplasia
Background Introduction
Intimal Hyperplasia (IH) is a common pathological response following vascular injury, particularly after procedures such as angioplasty or stent implantation. Although the use of Drug-Eluting Stents (DES) has significantly reduced restenosis rates, cardiovascular adverse events, including restenosis, still occur in 5% to 10% of patients. Therefore, identifying new therapeutic strategies to inhibit intimal hyperplasia is crucial.
Cyclooxygenase-2 (COX-2) is highly expressed in injured vessels, and its derivative Prostaglandin E2 (PGE2) is believed to play a significant role in promoting intimal hyperplasia. PGE2 exerts its effects through its receptor EP4, which is upregulated in injured vessels and promotes intimal hyperplasia in Vascular Smooth Muscle Cells (VSMCs). However, EP4 has been shown to inhibit intimal hyperplasia in endothelial cells. Thus, investigating the spatiotemporal expression of EP4 and its signaling mechanisms may provide new therapeutic targets for intimal hyperplasia.
Source of the Paper
This paper was authored by Shigekuni Okumura and colleagues and published in Cardiovascular Research, Volume 120, pages 2293-2306, in 2024. The research team included members from Tokyo Medical University, Oita University, Augusta University, Vanderbilt University Medical Center, and other institutions. The paper was received on November 8, 2023, accepted on August 6, 2024, and published online on September 22, 2024.
Research Process and Results
1. Spatiotemporal Expression of EP4 in Injured Vessels
The research team first generated EP4 reporter mice (ptger4-ires-nlslacz) and observed EP4 expression in a femoral artery injury model. The results showed that EP4 was transiently but significantly expressed in the proliferative neointima 2 weeks after injury, gradually decreasing thereafter. Through immunofluorescence staining, the researchers found that EP4 was primarily expressed in VSMCs, with no detectable expression in endothelial cells or monocytes/macrophages.
2. Role of EP4 Signaling in VSMCs
To investigate the role of EP4 in VSMCs, the research team generated VSMC-specific EP4-deficient mice (ptger4fl/+;sm22-cre) and EP4-overexpressing mice (ptger4-tg). The results showed that EP4-deficient mice exhibited significantly reduced intimal hyperplasia at 2 and 4 weeks post-injury, while EP4-overexpressing mice showed significantly increased intimal hyperplasia. These findings indicate that EP4 signaling in VSMCs promotes intimal hyperplasia.
3. EP4 Signaling Promotes Intimal Hyperplasia via Fibulin-1
The researchers further investigated the downstream mechanisms of EP4 signaling and found that EP4 stimulation increased Fibulin-1 mRNA and protein levels in VSMCs. Fibulin-1 is a secreted glycoprotein associated with the proliferation of various cell types. Through CRISPR/Cas9-mediated knockdown of Fibulin-1, the researchers found that EP4-mediated proliferation and migration were significantly attenuated in Fibulin-1-deficient VSMCs.
4. Fibulin-1 Promotes VSMC Proliferation and Migration via the TGF-β/Smad3 Signaling Pathway
The researchers discovered that Fibulin-1 promotes the production of active TGF-β1 by binding to Latent TGF-β Binding Protein 1 (LTBP1), thereby activating the TGF-β/Smad3 signaling pathway. Through inhibitor experiments, the researchers confirmed the critical role of the TGF-β receptor and Smad3 in Fibulin-1-mediated VSMC proliferation and migration.
5. ECM1 Cooperates with Fibulin-1 to Promote VSMC Proliferation
The researchers also found that EP4 stimulation upregulated the expression of Extracellular Matrix Protein 1 (ECM1). ECM1 cooperated with Fibulin-1 to further enhance VSMC proliferation and migration.
6. Fibulin-1 Deficiency Attenuates Intimal Hyperplasia
By generating VSMC-specific Fibulin-1-deficient mice (fbln1fl/fl;sm22-cre), the researchers found that Fibulin-1 deficiency significantly attenuated injury-induced intimal hyperplasia. This further confirmed the important role of Fibulin-1 in intimal hyperplasia.
7. EP4 Antagonist Attenuates Intimal Hyperplasia
Finally, through oral administration of an EP4 antagonist (CJ42794), the researchers found that the EP4 antagonist significantly attenuated injury-induced intimal hyperplasia. This suggests that EP4 antagonists may serve as a potential therapeutic strategy for intimal hyperplasia.
Conclusions and Significance
This study found that PGE2-EP4 signaling is activated in the proliferative neointima of injured vessels, and EP4 promotes VSMC proliferation and migration by upregulating Fibulin-1, thereby exacerbating intimal hyperplasia. The research also revealed that Fibulin-1 functions through the TGF-β/Smad3 signaling pathway and cooperates with ECM1 to promote VSMC proliferation and migration. These findings provide new insights into the molecular mechanisms of intimal hyperplasia and offer potential therapeutic targets.
Research Highlights
- Spatiotemporal Expression of EP4: For the first time, the spatiotemporal expression pattern of EP4 in injured vessels was revealed, clarifying its primary role in VSMCs.
- Mechanism of Fibulin-1: The molecular mechanism by which Fibulin-1 promotes VSMC proliferation and migration through the TGF-β/Smad3 signaling pathway was elucidated.
- Therapeutic Potential of EP4 Antagonists: The potential therapeutic value of EP4 antagonists in attenuating intimal hyperplasia was confirmed, providing new insights for clinical treatment.
Other Valuable Information
The research team also developed various genetically modified mouse models, including EP4 reporter mice, EP4-deficient mice, and Fibulin-1-deficient mice, which provided important tools for in-depth studies of EP4 and Fibulin-1 functions. Additionally, the study utilized CRISPR/Cas9 technology for gene knockdown experiments, further validating the critical role of Fibulin-1 in the EP4 signaling pathway.
Through this research, we have not only deepened our understanding of the molecular mechanisms of intimal hyperplasia but also provided a scientific basis for developing new therapeutic strategies. In the future, EP4 antagonists and Fibulin-1 regulation may become important approaches for treating vascular restenosis.