Proteomic Profiling of Early Secreted Proteins in Response to Lipopolysaccharide-Induced Vascular Endothelial Cell EA.hy926 Injury

Worawat Songjang; Nitchawat Paiyabhroma; Noppadon Jumroon; Arunya Jiraviriyakul; Nitirut Nernpermpisooth; Porrnthanate Seenak; Sarawut Kumphune; Siriwan Thaisakun; Narumon Phaonakrop; Sittiruk Roytrakul; Panyupa Pankhong

doi:10.3390/biomedicines11113065

Proteomic Profiling of Early Secreted Proteins in Response to Lipopolysaccharide-Induced Vascular Endothelial Cell EA.hy926 Injury

Biomedicines. 2023 Nov 15;11(11):3065. doi: 10.3390/biomedicines11113065.

Authors

Worawat Songjang^{1

2}, Nitchawat Paiyabhroma², Noppadon Jumroon^{1

2}, Arunya Jiraviriyakul^{1

2}, Nitirut Nernpermpisooth^{1

3}, Porrnthanate Seenak^{1

3}, Sarawut Kumphune^{1

4

5}, Siriwan Thaisakun⁶, Narumon Phaonakrop⁶, Sittiruk Roytrakul⁶, Panyupa Pankhong^{1

2}

Affiliations

¹ Integrative Biomedical Research Unit (IBRU), Faculty of Allied Health Sciences, Naresuan University, Phitsanulok 65000, Thailand.
² Department of Medical Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok 65000, Thailand.
³ Department of Cardio-Thoracic Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok 65000, Thailand.
⁴ Biomedical Engineering and Innovation Research Center, Chiang Mai University, Mueang Chiang Mai District, Chiang Mai 50200, Thailand.
⁵ Biomedical Engineering Institute (BMEI), Chiang Mai University, Chiang Mai 50200, Thailand.
⁶ Functional Proteomics Technology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathum Thani 12120, Thailand.

Abstract

Sepsis is a crucial public health problem with a high mortality rate caused by a dysregulated host immune response to infection. Vascular endothelial cell injury is an important hallmark of sepsis, which leads to multiple organ failure and death. Early biomarkers to diagnose sepsis may provide early intervention and reduce risk of death. Damage-associated molecular patterns (DAMPs) are host nuclear or cytoplasmic molecules released from cells following tissue damage. We postulated that DAMPs could potentially be a novel sepsis biomarker. We used an in vitro model to determine suitable protein-DAMPs biomarkers for early sepsis diagnosis. Low and high lipopolysaccharide (LPS) doses were used to stimulate the human umbilical vein endothelial cell line EA.hy926 for 24, 48, and 72 h. Results showed that cell viability was reduced in both dose-dependent and time-dependent manners. Cell injury was corroborated by a significant increase in lactate dehydrogenase (LDH) activity within 24 h in cell-conditioned medium. Secreted protein-DAMPs in the supernatant, collected at different time points within 24 h, were characterized using shotgun proteomics LC-MS/MS analysis. Results showed that there were 2233 proteins. Among these, 181 proteins from the LPS-stimulated EA.hy926 at 1, 12, and 24 h were significantly different from those of the control. Twelve proteins were up-regulated at all three time points. Furthermore, a potential interaction analysis of predominant DAMPs-related proteins using STITCH 5.0 revealed the following associations with pathways: response to stress; bacterium; and LPS (GO:0080134; 0009617; 0032496). Markedly, alpha-2-HS-glycoprotein (AHSG or fetuin-A) and lactotransferrin (LTF) potentially presented since the first hour of LPS stimulation, and were highly up-regulated at 24 h. Taken together, we reported proteomic profiling of vascular endothelial cell-specific DAMPs in response to early an in vitro LPS stimulation, suggesting that these early damage-response protein candidates could be novel early biomarkers associated with sepsis.

Keywords: DAMPs; biomarker; endothelial cell; lipopolysaccharide; proteomic; sepsis.

Grants and funding

R2565B088/The National Science, Research, and Innovation Fund (NSRF)