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Published on: November 26, 2018
Involvement of P2Y12 receptor in vascular smooth muscle inflammatory changes via MCP-1 upregulation and monocyte
Hiroshi Satonaka1, Daisuke Nagata2, Masao Takahashi2
1Department of Internal Medicine, Graduate School of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan; hsatonak-tky@umin.ac.jp.
Abstract:
Antiplatelet drugs, frequently used for cardiovascular events with thrombotic involvement, are also regarded as possible promising agents for cardiovascular primary prevention. The roles of P2Y12, an ADP receptor and the target of thienopyridine antiplatelet drugs, are not satisfactorily known in the vascular wall. We investigated the hypothesis that vascular smooth muscle cell (VSMC) P2Y12 is involved in vascular wall inflammatory changes by upregulating monocyte chemoattractant protein-1 (MCP-1) and promoting monocyte adhesion. ADP at 10(-5) M induced a 3.6 ± 0.3-fold upregulation of MCP-1 mRNA in cultured rat VSMCs, which was significantly inhibited by R-138727, the active metabolite of P2Y12 inhibitor prasugrel and siRNAs against P2Y12. ADP also induced MCP-1 protein upregulation, which was diminished by R-138727 and P2Y12 siRNAs. JNK (c-Jun NH2-terminal kinase) inhibition attenuated ADP-induced MCP-1 mRNA and protein upregulation. R-138727 and P2Y12 siRNAs inhibited ADP-induced JNK activation. The reactive oxygen species (ROS) inhibitors N-acetylcysteine (NAC), diphenyleneiodonium (DPI), and Tempol also diminished MCP-1 upregulation and JNK activation induced by ADP. ADP induced MCP-1 promoter activation, which was inhibited by R-138727 and P2Y12 siRNAs. Nuclear factor-κB (NF-κB) consensus sites in the MCP-1 promoter region were involved in this activation. ADP-induced NF-κB pathway activation, examined by a plasmid containing multiple NF-κB sites, was diminished by P2Y12 inhibition. For cellular function analysis, stimulation of VSMC with ADP increased subsequent THP-1 monocyte adhesion. P2Y12 siRNAs and CCR2 antagonism diminished this ADP-induced monocyte adhesion. These data suggested that ADP, via the VSMC P2Y12 receptor, induces vascular inflammatory changes by upregulating MCP-1 and promoting monocyte adhesion.
Insights
Vascular smooth muscle cell P2Y12 receptors mediate inflammation by increasing monocyte chemoattractant protein-1 (MCP-1) and monocyte adhesion. Inhibiting P2Y12 reduces these inflammatory responses, suggesting a role in cardiovascular primary prevention.
Area of Science:
- Cardiovascular Biology
- Molecular Pharmacology
- Immunology
Background:
- P2Y12 receptor antagonists are used for thrombotic cardiovascular events.
- The role of vascular smooth muscle cell (VSMC) P2Y12 in vascular inflammation is not well understood.
- Investigating P2Y12 in VSMCs may reveal new targets for cardiovascular disease prevention.
Purpose of the Study:
- To investigate the role of VSMC P2Y12 in vascular inflammation.
- To determine if P2Y12 signaling upregulates monocyte chemoattractant protein-1 (MCP-1) and promotes monocyte adhesion.
- To explore the signaling pathways involved, including JNK, ROS, and NF-κB.
Main Methods:
- Primary rat VSMCs were cultured and treated with ADP.
- P2Y12 expression was inhibited using R-138727 and siRNAs.
- MCP-1 mRNA and protein levels were quantified using RT-PCR and Western blotting.
- JNK activation, ROS production, and NF-κB pathway activation were assessed.
- Monocyte adhesion assays were performed using THP-1 cells.
- MCP-1 promoter activity was analyzed.
Main Results:
- ADP significantly upregulated MCP-1 mRNA and protein in VSMCs.
- P2Y12 inhibition (R-138727, siRNAs) attenuated ADP-induced MCP-1 upregulation and JNK activation.
- Inhibitors of JNK, ROS, and NF-κB diminished ADP-induced MCP-1 upregulation.
- ADP stimulation increased VSMC-monocyte adhesion, which was reduced by P2Y12 inhibition and CCR2 antagonism.
- ADP activated the MCP-1 promoter, involving NF-κB sites, and this was inhibited by P2Y12 blockade.
Conclusions:
- VSMC P2Y12 receptor activation by ADP promotes vascular inflammation.
- This process involves the upregulation of MCP-1 and subsequent monocyte adhesion via JNK, ROS, and NF-κB pathways.
- Targeting VSMC P2Y12 may offer a novel strategy for cardiovascular primary prevention.
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