Inhibition of the intracellular domain of Notch1 results in vascular endothelial cell dysfunction in sepsis

Tingyan Liu; Caiyan Zhang; Jiayun Ying; Yaodong Wang; Gangfeng Yan; Yufeng Zhou; Guoping Lu

doi:10.3389/fimmu.2023.1134556

Inhibition of the intracellular domain of Notch1 results in vascular endothelial cell dysfunction in sepsis

Front Immunol. 2023 May 2:14:1134556. doi: 10.3389/fimmu.2023.1134556. eCollection 2023.

Authors

Tingyan Liu¹, Caiyan Zhang¹, Jiayun Ying¹, Yaodong Wang¹, Gangfeng Yan¹, Yufeng Zhou^{2

3}, Guoping Lu¹

Affiliations

¹ Department of Critical Care Medicine, Children's Hospital of Fudan University, Shanghai, China.
² Institute of Pediatrics, Children's Hospital of Fudan University, National Children's Medical Center, and the Shanghai Key Laboratory of Medical Epigenetics, International Co-laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
³ National Health Commission (NHC) Key Laboratory of Neonatal Diseases, Fudan University, Shanghai, China.

Abstract

Background: Notch signaling is critical for regulating the function of vascular endothelial cells (ECs). However, the effect of the intracellular domain of Notch1 (NICD) on EC injury in sepsis remains unclear.

Methods: We established a cell model of vascular endothelial dysfunction and induced sepsis in a mouse model via lipopolysaccharide (LPS) injection and cecal ligation and puncture (CLP). Endothelial barrier function and expression of endothelial-related proteins were determined using CCK-8, permeability, flow cytometry, immunoblot, and immunoprecipitation assays. The effect of NICD inhibition or activation on endothelial barrier function was evaluated in vitro. Melatonin was used for NICD activation in sepsis mice. The survival rate, Evans blue dye of organs, vessel relaxation assay, immunohistochemistry, ELISA, immunoblot were used to explore the specific role of melatonin for sepsis induced vascular dysfunction in vivo.

Results: We found that LPS, interleukin 6, and serum collected from septic children could inhibit the expression of NICD and its downstream regulator Hes1, which impaired endothelial barrier function and led to EC apoptosis through the AKT pathway. Mechanistically, LPS decreased the stability of NICD by inhibiting the expression of a deubiquitylating enzyme, ubiquitin-specific proteases 8 (USP8). Melatonin, however, upregulated USP8 expression, thus maintaining the stability of NICD and Notch signaling, which ultimately reduced EC injury in our sepsis model and elevated the survival rate of septic mice.

Conclusions: We found a previously uncharacterized role of Notch1 in mediating vascular permeability during sepsis, and we showed that inhibition of NICD resulted in vascular EC dysfunction in sepsis, which was reversed by melatonin. Thus, the Notch1 signaling pathway is a potential target for the treatment of sepsis.

Keywords: LPS (lipopolysaccharide); NICD; melatonin; sepsis; vascular endothelial dysfunction.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Endothelial Cells / metabolism
Lipopolysaccharides
Melatonin* / pharmacology
Mice
Sepsis* / metabolism
Signal Transduction

Substances

Lipopolysaccharides
Melatonin
Notch1 protein, mouse

Grants and funding

This work was supported by grants from the National Key R&D Program of China (2021YFC2701800, 2021YFC2701805 to GL; 2021YFC2701802 to YZ), National Natural Science Foundation of China (82241038, 81974248 to YZ, 82202374 to CZ), and Shanghai Committee of Science and Technology (23ZR1407600, 21140902400, 21ZR1410000, 19ZR1406400 to YZ, 22ZR1408500 to CZ, 22Y11900500 to YW).