Trimethylamine-N-oxide affects cell type-specific pathways and networks in mouse aorta to promote atherosclerotic plaque vulnerability

Authors: CHENG, JENNY - University Of California (UCLA); CHENG, MICHAEL - University Of California (UCLA); SINHA, SATYESH - University Of California (UCLA); CELY, INGRID - University Of California (UCLA); GLADKIKH, SASHA - University Of California (UCLA); HAN, MAGGIE - University Of California (UCLA); ZHANG, GUANGLIN - University Of California (UCLA); ZHOU, ZHIQIANG - University Of California (UCLA); CHUGH, RUBANI - University Of California (UCLA); AHN, IN SOOK - University Of California (UCLA); DIAMANTE, GRACIEL - University Of California (UCLA); WANG, YU-CHEN - University Of California (UCLA); WANG, ZENENG - Cleveland Clinic; Bennett, Brian; CAI, HUA - University Of California (UCLA); ALLAYEE, HOOMAN - University Of California (UCLA); HAZEN, STANLEY - Cleveland Clinic; LUSIS, ALDONS - University Of California (UCLA); SHIH, DIANA - University Of California (UCLA); YANG, XIA - University Of California (UCLA)

Submitted to: Arteriosclerosis Thrombosis and Vascular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/22/2025
Publication Date: 10/1/2025
Citation: Cheng, J., Cheng, M., Sinha, S., Cely, I., Gladkikh, S., Han, M.T., Zhang, G., Zhou, Z., Chugh, R., Ahn, I., Diamante, G., Wang, Y., Wang, Z., Bennett, B.J., Cai, H., Allayee, H., Hazen, S., Lusis, A.J., Shih, D.M., Yang, X. 2025. Trimethylamine-N-oxide affects cell type-specific pathways and networks in mouse aorta to promote atherosclerotic plaque vulnerability. Arteriosclerosis Thrombosis and Vascular Biology. 45(10):1784-1798. https://doi.org/10.1161/ATVBAHA.125.323047.
DOI: https://doi.org/10.1161/ATVBAHA.125.323047

Interpretive Summary: This study is the first to examine the effects of Trimethylamine-N-oxide (TMAO) on disease-specific vascular cell types in atherosclerosis. Using single-cell RNA sequencing (scRNAseq) in Ldlr-/- mice, researchers found that TMAO upregulates apoptotic gene signatures while downregulating extracellular matrix (ECM) organization and collagen formation in vascular smooth muscle cells (vSMCs). Additionally, SMC-derived macrophages showed increased ECM degradation, which contributes to plaque instability. In vitro studies confirmed TMAO’s direct effects on vSMCs and macrophages. Network modeling further highlighted disrupted cell-cell communication and gene regulatory pathways linked to TMAO exposure. Importantly, TMAO-fed mice exhibited thinner fibrous caps, a key marker of plaque rupture risk. These findings suggest that TMAO promotes plaque instability by reducing collagen production and increasing its degradation, ultimately enhancing the likelihood of rupture. This study highlights TMAO’s role in atherosclerosis progression and underscores its potential impact on cardiovascular health.

Technical Abstract: Background Trimethylamine-N-oxide (TMAO) has been significantly linked to atherosclerosis via several mechanisms, but its direct effect on the atherosclerosis-prone vasculature remains unclear. The objective of this study was to characterize the cell type-dependent and independent effects of TMAO on key vascular cell types involved in atherosclerosis progression in vivo. Methods We performed single cell RNA-sequencing (scRNAseq) on aortic athero-prone regions of female Ldlr-/- mice fed control Chow, high-cholesterol (HC), or HC+TMAO diets for three months to identify which aortic cell types, differentially expressed genes, and biological pathways are affected by TMAO. We also modeled cell-cell communications and intracellular gene regulatory networks to identify gene networks perturbed by TMAOfeeding. Key genes and pathways were validated using primary human smooth muscle cells exposed to TMAO. Changes in the thickness of lesional fibrous caps in response to TMAO in female Ldlr-/- mice fed HC+TMAO versus HC diets were measured using transgelin immunostaining. Results Our scRNAseq analysis revealed that TMAO supplementation upregulated apoptotic gene signatures and downregulated extracellular matrix (ECM) organization and collagen formation genes in a subset of atherosclerosis-specific modulated vascular smooth muscle cells (vSMCs). We also identified “degradation of the ECM” as a top pathway for SMC-derived macrophage DEGs in response to TMAO. Network analyses support that macrophage-vSMC communication mediates ECM remodeling. Using human smooth muscle cells exposed to TMAO in vitro, we confirmed the direct effect of TMAO on regulating collagen and apoptotic genes. In agreement with the changes in these pathways that affect plaque stability, we observed a significant decrease in fibrous cap thickness in mice supplemented with TMAO. Conclusions Our results reveal the effects of TMAO on vSMCs to promote apoptosis and decrease ECM formation, and on macrophage-mediated ECM degradation in atherosclerotic lesions to in concert enhance plaque instability.