Paul-Lennard Mendez

Aims: Fibroblasts are mesenchymal cells in the healthy vascular adventitia. In atherosclerosis, single-cell sequencing datasets suggest fibroblasts are abundant in plaques. However, their identity, origin, and fate during plaque progression remain unclear, which we aim to unravel here. Methods and results: To robustly define fibroblast identity, origin, and fate, we employed meta-analyses of 54 single-cell RNA sequencing libraries, including murine smooth muscle cell (Myh11) and endothelial cell (EC) (Cdh5) lineage reporter mice with and without atherosclerosis; human control and atherosclerotic arteries; and murine adventitia and atherosclerotic plaques processed separately from low-density lipoprotein (LDL) receptor knockout (Ldlr−/−) mice. These meta-analyses showed that murine and human plaque fibroblast identity was robustly defined by Pdgfra, Pi16, Cygb, and Serpinf1 mRNA. Ninety-five percent of plaque fibroblasts do not derive from the Myh11 lineage, while no Cdh5-lineage-positive cells were present in the fibroblast cluster. We identified five murine arterial fibroblast subsets in atherosclerotic murine aorta: progenitor fibroblasts, matrix fibroblasts, inflammatory fibroblasts, an EC-like fibroblast subset, detected in both adventitia and plaques, and Col5a3+ fibroblasts, unique to the adventitia. We next studied fibroblast identity, origin, and fate using pseudotime analysis and Pdgfra-CreERT2/tdTomato lineage reporter mice (Pdgfra Lin+). Healthy Pdgfra Lin+ reporter mice showed predominant adventitial tdTomato expression, and infrequent medial and intimal Pdgfra Lin+ cells co-expressing MYH11 and PECAM1, respectively. The Pdgfra Lin+ plaque area increased with diet duration. Pdgfra Lin+ cells largely maintain fibroblast identity in the plaque, while <10% co-express SMC markers (MYH11, SM22α), or contribute to ACTA2+ cap cells. ECs gaining mesenchymal markers are transcriptionally distinct from Cdh5-lineage-negative fibroblasts gaining EC markers. Plaque-resident EC-like fibroblasts displayed a mesenchymal-to-endothelial transition transcriptome, which was induced in human primary fibroblasts in vitro by starvation, and dampened or reversed by IL1B, TGFB1, TGFB3, and oxidized LDL. Cross-species integration showed that all murine plaque fibroblasts were conserved in human atherosclerosis, with one additional subset partially resembling murine subsets, and three human-specific subsets. Importantly, human fibroblast subsets differentially correlated to human plaque traits, with EC-like fibroblasts correlating to plaque instability. Conclusion: Our results indicate that 95% of plaque-residing fibroblasts are Myh11 Lin− Plaque fibroblasts have a dual origin, predominantly adventitial Pdgfra Lin+ progenitor fibroblasts, with a minor contribution from medial Pdgfra Lin+ Myh11+ SMCs. Most plaque fibroblasts maintain fibroblast identity. Murine plaque fibroblast subsets were conserved in human atherosclerosis. EC-like fibroblasts are linked to human plaque instability. Intervening in progenitor-to-specific fibroblast transitions could present a new avenue to promote plaque stability in atherosclerosis.

Arterial aging is a major risk factor for cardiovascular disease and is associated with progressive changes in vascular structure and function, including arterial stiffening, reduced elasticity, extracellular matrix remodeling, chronic low‐grade inflammation, and accumulation of senescence‐associated cell states. Recent advances in single‐cell RNA sequencing (scRNA‐seq) have provided new opportunities to resolve the cellular heterogeneity underlying these age‐related alterations in the arterial wall. In this review, we summarize current single‐cell studies of arterial aging by focusing first on key phenotypic programs, including cellular senescence, extracellular matrix remodeling, inflammaging, and altered intercellular communication, and then discuss how these programs are reflected in endothelial cells, smooth muscle cells, fibroblasts, and immune cells. Across studies, aging is recurrently associated with endothelial dysfunction, smooth muscle cell phenotypic modulation, fibroblast‐related matrix remodeling, and immune activation, although the degree of conservation varies depending on species, vascular bed, sex, and disease context. We further discuss emerging evidence that vascular aging involves not only cell‐intrinsic transcriptional changes but also alterations in communication networks across the arterial wall. Although current single‐cell studies have substantially improved our understanding of arterial aging, important limitations remain, including inconsistent cell‐state annotation across studies, incomplete functional validation, and limited spatial and epigenetic resolution. Future integration of cross‐species analyses with spatial transcriptomics, single‐cell epigenomic approaches, and functional studies will help refine the cellular framework of arterial aging and improve its translational relevance.

Endothelial-to-mesenchymal transition (EndMT) contributes to vascular remodeling and endothelial dysfunction in diseases such as atherosclerosis and pulmonary arterial hypertension. This process describes a gradual shift in which endothelial cells (ECs) lose expression of classical EC markers and acquire mesenchymal-like gene expression patterns and phenotypic traits. Although SMAD transcription factors are key regulators of EndMT, driven by mechanical cues from disturbed flow that activate BMP or TGFβ signaling, their weak DNA-binding affinity invoked the need for additional transcriptional co-factors to specify gene targets under pathophysiological flow conditions. Here, we identified FOXO1 as a transcriptional co-factor that cooperates with SMADs to regulate mesenchymal gene expression in ECs exposed to irregular flow. FOXO1 and SMADs co-occupy genomic loci and form transcriptional complexes. Although SMAD DNA binding is FOXO1-independent, FOXO1 activity is required for full induction of SMAD target genes. Notably, FOXO1 DNA-binding activity is essential for robust activation of EndMT-associated genes, as both pharmacological inhibition and siRNA-mediated depletion of FOXO1 attenuate SMAD target gene expression. Functionally, combined targeting of FOXO1 and SMAD signaling rescues EndMT-associated endothelial dysfunction in carotid arteries of mice. Our results describe FOXO1 as key integrator of SMAD-mediated EndMT signaling, linking it to pathogenic SMAD activation in disease contexts.

While somatic variants are well-characterized drivers of tumor evolution, their influence on cellular fitness in nonmalignant contexts remains understudied. We identified a mosaic synonymous variant (m.7076A > G) in the mitochondrial DNA (mtDNA)-encoded cytochrome c-oxidase subunit 1 (MT-CO1, p.Gly391=), present at homoplasmy in 47% of immune cells from a healthy donor. Single-cell multiomics revealed strong, lineage-specific selection against the m.7076G allele in CD8 ⁺ effector memory T cells, but not other T cell subsets, mirroring patterns of purifying selection of pathogenic mtDNA alleles. The limited anticodon diversity of mitochondrial tRNAs forces m.7076G translation to rely on wobble pairing, unlike the Watson–Crick–Franklin pairing used for m.7076A. Mitochondrial ribosome profiling confirmed stalled translation of the m.7076G allele. Functional analyses demonstrated that the elevated translational and metabolic demands of short-lived effector T cells (SLECs) amplify dependence on MT-CO1, driving this selective pressure. These findings suggest that synonymous variants can alter codon syntax, impacting mitochondrial physiology in a cell type–specific manner.