Trimethylamine-N-Oxide Affects Cell Type-Specific Pathways and Networks in Mouse Aorta to Promote Atherosclerotic Plaque Vulnerability.

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 on aortic athero-prone regions of female Ldlr^-/- mice fed normal laboratory, high-cholesterol, or high-cholesterol+TMAO diets for 3 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 TMAO feeding. Key genes and pathways were validated using human vascular smooth muscle cells (vSMCs) exposed to TMAO. Changes in fibrous cap thickness, macrophage content, and collagen deposition in response to TMAO were measured with immunostaining and histology and quantified.

Results: Our single-cell RNA-sequencing 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 vSMCs. We also identified degradation of the ECM as a top pathway for vSMC-derived macrophage differentially expressed genes in response to TMAO. Network analyses supported 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 and collagen deposition 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 to, in concert enhance atherosclerotic plaque instability.