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Protective effects of acetylcholine on hypoxia-induced endothelial-to-mesenchymal transition in human cardiac microvascular endothelial cells

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Abstract

Endothelial-to-mesenchymal transition (EndMT) has been reported as a key factor in myocardial fibrosis. Acetylcholine (ACh), a neurotransmitter of the vagus nerve, has been confirmed to exert cardio-protective properties with unclear mechanisms. In this study, the specific markers of cell injury, EndMT, inflammation, and autophagy were measured. We found that treatment with ACh prevented hypoxia-induced cell viability reduction and apoptosis in human cardiac microvascular endothelial cells (HCMECs). Additionally, our results indicate that pre-treatment with ACh significantly suppresses hypoxia-induced EndMT and NF-κB activation in HCMECs. ACh also reduced hypoxia-inducible factor (HIF)-1ɑ protein levels under hypoxia. Knock down of HIF-1ɑ enhanced the inhibitory effect of ACh on NF-κB activation. The NF-κB-specific small molecule inhibitor BAY 11-7082, prostaglandin E2, and LY294002 prevented hypoxia-induced EndMT. Moreover, our data show that hypoxia triggers autophagy in HCMECs, and ACh significantly upregulates autophagy activity. Pre-treatment of HCMECs with 3-methyladenine or chloroquine partially reversed ACh-induced EndMT inhibition. These results suggest that ACh may confer protection against hypoxia-induced EndMT through the inhibition of NF-κB and the induction of autophagy.

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Data availability

All data generated or analyzed during this study are included in this published article.

Abbreviations

α-SMA:

α-Smooth muscle actin

Ach:

Acetylcholine

AMI:

Acute myocardial infarction

ECM:

Extracellular matrix

EMT:

Epithelial-to-mesenchymal transition

EndMT:

Endothelial-to-mesenchymal transition

HCAECs:

Human coronary artery endothelial cells

HCMECs:

Human cardiac microvascular endothelial cells

HUVECs:

Human umbilical vein endothelial cells

MF:

Myocardial fibrosis

PBS:

Phosphate buffered saline

PECAM-1:

Platelet endothelial cell adhesion molecule-1

TEM:

Transmission electron microscopy

VE-cad:

Vascular endothelial cadherin

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 81860052, 81871113).

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Author notes

  1. Zhiyang Li, Xuelian Li, and Yeqian Zhu have contributed equally to this study.

Authors and Affiliations

  1. Grade 2016 Class 2, The First School of Clinical Medicine, Nanjing Medical University, Nanjing, 211166, China

    Zhiyang Li

  2. Department of Cardiology, Qingdao Municipal Hospital, Nanjing Medical University, Qingdao, 266011, China

    Xuelian Li & Bingong Li

  3. Department of Cardiology, The First Affiliated Hospital of Nanchang University, Nanchang, 330006, China

    Xuelian Li & Bingong Li

  4. The Section of Pacing and Electrophysiology, Division of Cardiology, The First Affiliated Hospital with Nanjing Medical University, Nanjing, 211166, China

    Yeqian Zhu, Qiushi Chen & Fengxiang Zhang

  5. Section of Pacing and Electrophysiology, Division of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Guangzhou Road 300, Nanjing, 210029, People’s Republic of China

    Fengxiang Zhang

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  1. Zhiyang Li
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All the authors participated in the design, interpretation of the data, drafting and review of the manuscript.

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Correspondence to Fengxiang Zhang.

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Li, Z., Li, X., Zhu, Y. et al. Protective effects of acetylcholine on hypoxia-induced endothelial-to-mesenchymal transition in human cardiac microvascular endothelial cells. Mol Cell Biochem 473, 101–110 (2020). https://doi.org/10.1007/s11010-020-03811-w

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