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DLK1 promoted ischemic angiogenesis through notch1 signaling in endothelial progenitor cells

Acta Pharmacologica Sinica (2024)Cite this article

Abstract

Delta like non-canonical Notch ligand 1 (DLK1), as a member of epidermal growth factor-like family, plays a critical role in somatic growth, tissue development and possibly tissue renewal. Though previous studies had indicated that DLK1 contributed to adipogenesis and myogenesis, it’s still controversial whether DLK1 affects angiogenesis and how it interacts with Notch signaling with numerous conflicting reports from different models. Based on our preliminary finding that DLK1 expression was up-regulated in mice ischemic gastrocnemius and in the border zone of infarcted myocardium, we administered either recombinant DLK1 (rDLK1) or PBS in C57BL/6 mice after establishment of hindlimb ischemia (HLI) and myocardial infarction (MI), respectively. Exogenous rDLK1 administration significantly improved both blood perfusion of mice ischemic hindlimbs and muscle motor function on the 3rd, 7th day after HLI, by promoting neovascularization. Similar effect on neovascularization was verified in mice on the 28th day after MI as well as improvement of cardiac failure. Correspondingly, the number of CD34+KDR+ cells, indicated as endothelial progenitor cells (EPCs), was significantly in mice ischemic gastrocnemius by rDLK1 administration, which was abrogated by DAPT as the specific inhibitor of Notch intracellular domain (NICD). Furthermore, bone marrow mononuclear cells were obtained from C57BL/6 mice and differentiated to EPCs ex vivo. Incubation with rDLK1 triggered Notch1 mRNA and NICD protein expressions in EPCs as exposed to hypoxia and serum deprivation, promoting EPCs proliferation, migration, anti-apoptosis and tube formation. Otherwise, rDLK1 incubation significantly decreased intracellular and mitochondrial reactive oxygen species, increased ATP content and mitochondrial membrane potential, downregulated short isoform of OPA-1 expression whereas upregulated mitofusin (-1, -2) expression in EPCs by Notch1 signaling, which were all abrogated by DAPT. In summary, the present study unveils the pro-angiogenesis and its mechanism of rDLK1 through activation of Notch1 signaling in endothelial progenitor cells.

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Fig. 1: Administration of exogenous rDLK1 promoted hindlimb perfusion recovery and neovascularization in HLI mice.
Fig. 2: Administration of exogenous rDLK1 triggered EPCs mobilization and infiltration into ischemic gastrocnemius.
Fig. 3: Perfusion recovery and neovascularization by exogenous rDLK1 were dependent on Notch1 signaling.
Fig. 4: Notch1 signaling was involved in mobilization and recruitment of EPCs by rDLK1.
Fig. 5: Administration of exogenous rDLK1 improved post-MI cardiac function by Notch1 signaling-dependent neovascularization.
Fig. 6: Exogenous rDLK1 altered biological functions of EPCs by Notch1 signaling.
Fig. 7: Exogenous rDLK1 affected mitochondria function and dynamics of EPCs by Notch1 signaling.
Fig. 8: Schematic diagram of the role and underlying mechanism of DLK1 in ischemic injury.

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All data and material during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

This study was supported by the National Natural Science Foundation of China (82270498) and National Health Commission, key program of science and technology of the Medical and Health of Zhejiang Province (WKJ-ZJ-2028).

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  1. These authors contributed equally: Ya-yu You, Ning Zhang

Authors and Affiliations

  1. Department of Cardiology, State Key Laboratory of Transvascular Implantation Devices, Cardiovascular Key Laboratory of Zhejiang Province, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China

    Ya-yu You, Zhuo Wang, Zhe-hui Yin, Qin-yi Bao, Shu-xin Lei & Xiao-jie Xie

  2. Department of Cardiology, Affiliated Hangzhou First People’s Hospital, School of Medicine, Westlake University, Hangzhou, 310009, China

    Ning Zhang

  3. International Institutes of Medicine, Fourth Affiliated Hospital, Zhejiang University School of Medicine, Yiwu, 322000, China

    Zhuo Wang

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Contributions

YYY and NZ conceived and designed the experiments. YYY, ZW and ZHY performed the experiments. YYY, QYB and SXL analyzed the data. YYY wrote the paper. XJX reviewed and revised the paper. All authors contributed to the article and approved the submitted version.

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Correspondence to Xiao-jie Xie.

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The authors declare no competing interests.

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The use of clinical samples was approved by the Institutional Ethics Committee of Second Affiliated Hospital of Zhejiang University. All animal experiments were approved by Zhejiang University Institutional Animal Care and Use Committee (approval No. 2023-1969). All methods and experimental protocols were carried out in accordance with the guidelines and regulations of the Care and Use of Laboratory Animals of Zhejiang University.

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You, Yy., Zhang, N., Wang, Z. et al. DLK1 promoted ischemic angiogenesis through notch1 signaling in endothelial progenitor cells. Acta Pharmacol Sin (2024). https://doi.org/10.1038/s41401-024-01346-0

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