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

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.