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Comparative analysis of pathways induced by TMAO and TNF-α in human microvascular endothelial cells
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  • Published: 03 April 2026

Comparative analysis of pathways induced by TMAO and TNF-α in human microvascular endothelial cells

  • Meyammai Shanmugham1,2,
  • Arun George Devasia1,3,
  • Gokce Oguz3,
  • Adaikalavan Ramasamy2,3,
  • Zen Zhi Yan Lim2,
  • Sophie Bellanger2 &
  • …
  • Chen Huei Leo4 

Scientific Reports , Article number:  (2026) Cite this article

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We are providing an unedited version of this manuscript to give early access to its findings. Before final publication, the manuscript will undergo further editing. Please note there may be errors present which affect the content, and all legal disclaimers apply.

Subjects

  • Biological techniques
  • Biotechnology
  • Cell biology
  • Computational biology and bioinformatics
  • Molecular biology
  • Next-generation sequencing
  • Sequencing

Abstract

Endothelial dysfunction is a systemic disorder that triggers vascular alterations, characterised by a reduction in nitric oxide (NO) synthesis and/or a defective vasodilatory response. Trimethylamine-N-oxide (TMAO) is a gut microbiota-derived dietary metabolite, while tumour necrosis factor alpha (TNF-α) functions as a pro-inflammatory cytokine. Both are known to induce inflammation, metabolic modulations, and endothelial dysfunction, contributing to cardiometabolic diseases. Nevertheless, the comparative effects of TMAO and TNF-α on inflammation and metabolic modulations have yet to be investigated. Here, using bulk RNA-sequencing (RNA-seq), real-time quantitative polymerase chain reaction (RT-qPCR) and interleukins/chemokines multiplex assays, we demonstrate significantly higher levels of inflammation, with a stronger cytokine response and activation of type I and II interferons, following TNF-α treatment compared to TMAO treatment in human dermal microvascular endothelial cells (HMEC-1). In addition, TNF-α upregulates the disassembly of the extracellular matrix (ECM), while ECM-related genes and pathways are either not modulated or down-regulated after TMAO treatment. Intriguingly, TMAO specifically induces a shift in energy metabolism (upregulation of OXPHOS (oxidative phosphorylation)), while TNF-α rather modulates lipid metabolism. In conclusion, this study reveals common pathways, but also key differences in molecular processes activated by TNF-α versus TMAO. These data are essential for identifying the most suitable in vitro human cellular models to study inflammation, as well as to discover novel targeted therapeutics to alleviate cardiometabolic conditions.

Data availability

The datasets generated and/or analysed during the current study are available in the Gene Expression Omnibus (GEO) repository, GSE235204 and in Sequence Read Archive (SRA) under accession number PRJNA1233874.

Abbreviations

ADAM:

A disintegrin and metalloproteinase

CVDs:

Cardiovascular diseases

CYP7A1:

Cholesterol 7α-hydroxylase

COL:

Collagens

COX:

Cyclooxygenase

ECM:

Extracellular matrix

FDR:

False discovery rate

FBS:

Fetal bovine serum

FLN:

Filamins

FLT:

Fms-related tyrosine kinases

GEO:

Gene expression omnibus

GO BPs:

Gene ontology biological processes

GSEA:

Gene set enrichment analysis

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

HMEC-1:

Human microvascular endothelial cells

HPKs:

Human primary keratinocytes

HUVEC:

Human umbilical vein endothelial cells

ITG:

Integrins

KEGG:

Kyoto encyclopaedia of genes and genomes

LAM:

Laminins

mTNF:

Membrane bound form of TNF

NO:

Nitric oxide

NF-κB:

Nuclear factor kappa B

ORA:

Over-representation analysis

OXPHOS:

Oxidative phosphorylation

PDGF:

Platelet-derived growth factors

PCA:

Principal component analysis

ROS:

Reactive oxygen species

RT-qPCR:

Real-time quantitative polymerase chain reaction

RNA-seq:

RNA-sequencing

sTNF:

Soluble TNF

SRA:

Sequence Read Archive

SEM:

Standard error of the mean

TACE:

TNF-α converting enzyme

TMAO:

Trimethylamine-N-oxide

TNF-α:

Tumour necrosis factor alpha

UQCR:

Ubiquinol-cytochrome c reductase

UPR:

Unfolded protein response

V-ATPase:

Vacuolar H+-ATPase

VST:

Variance stabilizing transformation

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Acknowledgements

MS was a recipient of the SUTD Ph.D. Scholarship. The authors thank Associate Professor Xiaogang Liu, Associate Professor Desmond Loke and Dr Christina Tan for their support and guidance in this study.

Funding

The research was partially funded by SUTD Start-up Research Grant (SRG-SMT-2020–156), SUTD-ZJU Grant (ZJUVP2000102), SUTD Kickstarter Initiative (SKI_2021_02_05), and the Agency for Science, Technology and Research (A*STAR). The sponsors have no role in the study design, collection, analysis, and interpretation of data, or writing of the report and in the decision to submit the article for publication.

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  1. Science, Math & Technology, Singapore University of Technology & Design, 8 Somapah Road, Singapore, 487372, Republic of Singapore

    Meyammai Shanmugham & Arun George Devasia

  2. Skin Research Institute of Singapore (SRIS), Agency for Science, Technology and Research (A*STAR), 8 A Biomedical Grove, Immunos, Singapore, #06-06, 138648, Republic of Singapore

    Meyammai Shanmugham, Adaikalavan Ramasamy, Zen Zhi Yan Lim & Sophie Bellanger

  3. Genome Institute of Singapore (GIS), Agency for Science Technology and Research (A*STAR), 60 Biopolis Street, Genome, 138672, Republic of Singapore

    Arun George Devasia, Gokce Oguz & Adaikalavan Ramasamy

  4. Department of Biomedical Engineering, College of Design & Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore, 117576, Republic of Singapore

    Chen Huei Leo

Authors
  1. Meyammai Shanmugham
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  2. Arun George Devasia
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  3. Gokce Oguz
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  4. Adaikalavan Ramasamy
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  6. Sophie Bellanger
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Contributions

Meyammai Shanmugham: Investigation, conceptualization, methodology, formal analysis, visualisation, writing- original draft preparationArun George Devasia: Methodology, formal analysis, writing- review & editingGokce Oguz: Methodology, formal analysis, visualisation, conceptualization, writing - review & editingAdaikalavan Ramasamy: Supervision, methodology, formal analysis, visualisation, conceptualization, writing - review & editingZen Zhi Yan Lim: InvestigationSophie Bellanger: Supervision, writing - review & editingChen Huei Leo: Supervision, Conceptualization, project administration, methodology, formal analysis, visualisation, funding acquisition, writing - review & editing.

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Correspondence to Chen Huei Leo.

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Shanmugham, M., Devasia, A.G., Oguz, G. et al. Comparative analysis of pathways induced by TMAO and TNF-α in human microvascular endothelial cells. Sci Rep (2026). https://doi.org/10.1038/s41598-026-46466-1

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  • Received: 24 February 2025

  • Accepted: 26 March 2026

  • Published: 03 April 2026

  • DOI: https://doi.org/10.1038/s41598-026-46466-1

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Keywords

  • TMAO
  • TNF-α
  • Inflammation
  • Endothelial dysfunction
  • Cardiometabolic diseases
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