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Circulating small extracellular vesicles promote proliferation and migration of vascular smooth muscle cells via AXL and MerTK activation

Abstract

The proliferation and migration of vascular smooth muscle cells (VSMCs) after vascular injury lead to neointimal hyperplasia, thus aggravating vascular diseases. However, the molecular mechanisms underlying neointima formation are not fully elucidated. Extracellular vesicles (EVs) are mediators of various intercellular communications. The potential of EVs as regulators in cardiovascular diseases has raised significant interest. In the current study we investigated the role of circulating small extracellular vesicles (csEVs), the most abundant EVs (1010 EVs/mL serum) in VSMC functions. csEVs were prepared from bovine, porcine or rat serum. We showed that incubation with csEVs (0.5 × 1010−2 × 1010) dose-dependently enhanced the proliferation and migration of VSMCs via the membrane phosphatidylserine (PS). In rats with ligation of right carotid artery, we demonstrated that application of csEVs in the ligated vessels aggravated neointima formation via interaction of membrane PS with injury. Furthermore, incubation with csEVs markedly enhanced the phosphorylation of AXL and MerTK in VSMCs. Pretreatment with BSM777607 (pan-TAM inhibitor), bemcentinib (AXL inhibitor) or UNC2250 (MerTK inhibitor) blocked csEV-induced proliferation and migration of VSMCs. We revealed that csEV-activated AXL and MerTK shared the downstream signaling pathways of Akt, extracellular signal-regulated kinase (ERK) and focal adhesion kinase (FAK) that mediated the effects of csEVs. We also found that csEVs increased the expression of AXL through activation of transcription factor YAP, which might constitute an AXL-positive feedback loop to amplify the signals. Finally, we demonstrated that dual inhibition of AXL/MerTK by ONO-7475 (0.1 µM) effectively hindered csEV-mediated proliferation and migration of VSMCs in ex vivo mouse aorta injury model. Based on these results, we propose an essential role for csEVs in proliferation and migration of VSMCs and highlight the feasibility of dual AXL/MerTK inhibitors in the treatment of vascular diseases.

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Fig. 1: csEV-induced proliferation and migration of VSMCs.
Fig. 2: Involvement of phosphatidylserine membrane structure in csEV-VSMC interaction.
Fig. 3: AXL/MerTK-dependent proliferation and migration of VSMC.
Fig. 4: Role of csEV-induced Akt/mTOR/S6K and ERK activation in the proliferation and migration of VSMC.
Fig. 5: Involvement of FAK activation in csEV-induced VSMC migration.
Fig. 6: YAP-dependent AXL upregulation in csEV-treated VSMC.
Fig. 7: Role of FAK in csEV-mediated YAP activation.
Fig. 8: Dual inhibition of AXL and MerTK completely blocks csEV-induced proliferation and migration of VSMCs.

Data availability

Supplementary information is available at the website of Acta Pharmacologica Sinica.

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Acknowledgements

This research was funded by the National Research Foundation of Korea (NRF) grants funded by the Korean Government; 2021R1A4A1021787 and 2022M3A9B6017654. Graphical representation was created with ©biorender.com.

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Contributions

YJL: conceptualization, investigation, validation, writing - original draft. MP: writing - review and editing. HYK: writing - review and editing. JKK: resources. WKK: methodology. SCL: methodology. KWK: conceptualization, writing - review and editing, funding acquisition, supervision.

Corresponding author

Correspondence to Keon Wook Kang.

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The authors declare no competing interests.

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Lee, Y.J., Park, M., Kim, H.Y. et al. Circulating small extracellular vesicles promote proliferation and migration of vascular smooth muscle cells via AXL and MerTK activation. Acta Pharmacol Sin (2022). https://doi.org/10.1038/s41401-022-01029-8

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  • DOI: https://doi.org/10.1038/s41401-022-01029-8

Keywords

  • vascular smooth muscle cells
  • circulating small extracellular vesicles
  • neointimal hyperplasia
  • phosphatidylserine
  • AXL
  • MerTK
  • BSM777607
  • bemcentinib
  • UNC2250
  • ONO-7475

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