A new study published in Engineering uses single-cell RNA sequencing (scRNA-seq) to unpack how phytosterol oxidation products (POPs), common dietary lipid derivatives, alter vascular cell heterogeneity and drive atherosclerosis (AS) through defined inflammatory signaling pathways. Atherosclerosis is a chronic inflammatory cardiovascular disorder where macrophage function shapes plaque formation and progression, yet prior research has not fully clarified how food-derived POPs reshape aortic cellular profiles or the molecular cascades linking POP exposure to vascular inflammation.

A research team from Zhejiang University built a single-cell transcriptomic atlas of aortic tissue from ApoE⁻/⁻ mice fed standard atherogenic diets with or without supplementary POPs, resolving five primary aortic cell lineages: fibroblasts, myeloid cells, lymphocytes, smooth muscle cells and endothelial cells. Cross-group comparison of cell-cell communication networks revealed POP treatment rewired intercellular signaling across plaque microenvironments, with myeloid cells emerging as central coordinators of inflammatory cross-talk between distinct vascular cell populations. Further subclustering of myeloid subsets identified four macrophage subpopulations, among which M1-like pro-inflammatory macrophages showed elevated representation in POP-fed mice, marked by enriched expression of canonical pro-inflammatory genes including Nlrp3, Tnf and Ccl3. Pseudotime trajectory and RNA velocity analyses mapped monocyte differentiation toward M1-like macrophage states under POP exposure, highlighting Il1r1 as a top dynamic driver gene governing this inflammatory cell fate shift.

(a) Workflow for single-cell RNA transcriptome experiments. (b) t-SNE visualization displayed (i) 20 minor and (ii) five major clusters in (iii) two groups. (c) Dot plot of five major cell markers. (d) Heatmap of differentially expressed genes in each subpopulation. The proportions of major clusters in the Con and POP groups were shown in (e) a stacked bar plot and (f) a three dimensional pie chart. (g) Ligand–receptor pair numbers per cell type in Con and POP groups. (h) Interaction intensity between myeloid cell and other cell types in the Con and POP groups. (i) Bubble chart depicting markedly elevated ligand–receptor interactions between source and target cells, color-coded by experimental group. The selected ligand–receptor pairs orchestrating myeloid-to-nonmyeloid cellular crosstalk in the Con and POP groups.

Gene enrichment and single-cell regulatory network analysis pinpointed the TLR4 signaling pathway and transcription factor IRF5 as dominant regulators of M1-like macrophage transcriptional activity in POP-treated samples. To validate this axis, the team ran in vitro assays on bone marrow-derived macrophages (BMDMs) and RAW264.7 macrophage cell lines stimulated with oxidized low-density lipoprotein (ox-LDL), the primary lipid accumulated within atherosclerotic plaques. 7-ketositosterol (7-KS), the most abundant POP found in processed foods, dose-dependently amplified secretion of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 while upregulating M1 surface markers iNOS and CD86, with no measurable change in anti-inflammatory mediator IL-10 release. Western blot and immunofluorescence assays confirmed 7-KS elevated protein levels of TLR4, MyD88 and TRAF6, and boosted IRF5 nuclear translocation to activate downstream inflammatory gene transcription. When cells were pre-treated with TLR4 inhibitor TLR4-IN-C34 or IRF5 inhibitor YE6144, the pro-inflammatory and M1-polarizing effects of 7-KS were largely eliminated, confirming the TLR4–IRF5 axis as an indispensable mediator of 7-KS activity.

To translate mouse-derived findings to human vascular pathology, researchers integrated four public human arterial scRNA-seq datasets covering healthy aortic tissue and advanced atherosclerotic carotid lesions. Across over 73,000 human vascular cells, inflammatory macrophage populations carrying matching POP-associated molecular signatures were significantly expanded in patient plaque samples, with GSEA confirming consistent enrichment of TLR signaling, MyD88-dependent TLR cascades and inflammatory response gene sets in human lesion macrophages. SCENIC transcription factor profiling detected heightened IRF5 regulon activity in human inflammatory macrophages, mirroring observations in murine M1-like macrophage populations.

The work establishes a single-cell framework for evaluating dietary component bioactivity and provides a conserved molecular signature linking dietary POP intake to vascular inflammation across mouse and human atherosclerotic tissue, while identifying potential molecular targets for mitigating diet-driven atherosclerotic progression.

Source – Higher Education Press