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PDK1 upregulates PINK1-mediated pulmonary endothelial cell mitophagy during hypoxia-induced pulmonary vascular remodeling

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Abstract

Background

Hypoxic pulmonary hypertension (HPH) is a complication of lung diseases with pulmonary vascular remodeling, although the underlying molecular mechanisms have not been fully elucidated. This study investigated the underlying molecular events by using a rat HPH model and primary pulmonary microvascular endothelial cells (PMVECs).

Methods and results

This study first established a rat HPH model and cultured PMVECs for transmission electron microscopic analysis and manipulation of 3-phosphoinositide-dependent protein kinase 1 (PDK1) or phosphatase and tensin homolog-induced kinase 1 (PINK1) expression in vitro. After that, the cell viability was assessed and the expression of different proteins was assayed using cell viability and western blot assays, respectively. Reactive oxygen species production, apoptosis, NLR family pyrin domain containing 3 (NLRP3) expression, and the levels of interleukin (IL)-1β, IL-6, and IL-8 were also assessed, while the interaction of PDK1 and PINK1 was determined using co-immunoprecipitation/western blot assays. Hypoxia induced mitophagy in the PMVECs and upregulated PINK1/Parkin expression, whereas knockdown of PINK1 expression under hypoxic conditions inhibited cell proliferation but induced endothelial cell apoptosis in vitro, decreased reactive oxygen species production and NLRP3 expression, and reduced the levels of inflammatory factors in PMVECs. However, hypoxia induced PDK1 expression, whereas knockdown of PDK1 downregulated PINK1 expression. Furthermore, treatment of the model rats with the PDK1 inhibitor dichloroacetate (DCA) was able to decrease PINK1 expression. In addition, the PDK1 and PINK1 proteins could interact with each other in the mitochondria of PMVECs to regulate the cell viability.

Conclusions

This study revealed that PDK1 induced PMVEC proliferation but inhibited their apoptosis to participate in pulmonary vascular remodeling, ultimately leading to HPH through regulation of PINK1-mediated mitophagy signaling. Therefore, PINK1 is a novel therapeutic target for the control of HPH.

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Data availability

The data that support the findings of this study are not publicly available due to none of the data types requiring uploading to a public repository but are available from the corresponding author upon reasonable request.

Abbreviations

CCK-8:

cell counting kit-8

DCA:

dichloroacetate

HPH:

hypoxic pulmonary hypertension

IL:

interleukin

NLRP3:

NLR family pyrin domain containing 3

PDK:

pyruvate dehydrogenase kinase

PINK1:

phosphatase and tensin homolog-induced kinase 1

PMVECs:

pulmonary microvascular endothelial cells

ROS:

reactive oxygen species

siRNA:

small interfering RNA

VEGF:

vascular endothelial growth factor

VEGFR:

vascular endothelial growth factor receptor

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Acknowledgements

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Funding

This study was supported in part by grants from the National Natural Science Foundation of China (#81571839 and #82072102).

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Author notes

  1. Jing Wang and Yue Zhang have contributed equally to this work.

Authors and Affiliations

  1. School of Life Sciences, Northwest University, Xi’an, 710069, Shaanxi, China

    Jing Wang, Yue Zhang & Zhi Chao Li

  2. Department of Physiology and Pathophysiology, Air Force Medical University, 169 Changle West Road, Xi’an, 710032, Shaanxi, China

    Ying Luo, Man Ling Liu, Wen Niu, Zhi Chao Li & Bo Zhang

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Contributions

BZ was mainly responsible for the design of the experiment and the writing of the article. JW was responsible for establishing the animal model, cell culture, the electron microscopy assay, and statistical analysis. YZ was responsible for the RT‑qPCR and western blot assays. YL was responsible for cell transfection, proliferation, and apoptosis investigations. ML was responsible for the CoIP assay. WN was responsible for reviewing and editing the manuscript. ZL was responsible for the design of the experiment. All authors read and approved the final version of the manuscript.

Corresponding authors

Correspondence to Zhi Chao Li or Bo Zhang.

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The authors declare that they have no competing interests.

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This study was approved by the Animal Use and Care Committee for Research and Education of The Fourth Military Medical University (Xi’an, China), and all experimental procedures followed the Guidelines of the Care and Use of Laboratory Animals issued by the Chinese Council on Animal Research. The study is reported in accordance with ARRIVE guidelines.

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Wang, J., Zhang, Y., Luo, Y. et al. PDK1 upregulates PINK1-mediated pulmonary endothelial cell mitophagy during hypoxia-induced pulmonary vascular remodeling. Mol Biol Rep 50, 5585–5596 (2023). https://doi.org/10.1007/s11033-023-08428-y

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