Targeting Modulated Vascular Smooth Muscle Cells in Atherosclerosis via FAP-Directed Immunotherapy
Junedh AmruteiD, In‐Hyuk JungiD, Tracy YamawakiiD, Wen-Ling LiniD, Andrea L. BredemeyeriD, Johanna DiekmanniD, Sikander HayatiD, Xianglong ZhangiD, Devin L. WakefieldiD, Xin LuoiD, Sidrah Maryam, Gyu Seong HeoiD, Steven Hoseong YangiD, Chang Jie Mick LeeiD, Chen WangiD, Caroline ChouiD, Christoph KuppeiD, Kevin D. CookiD, Atilla Kovacs, Vishnu ChintalgattuiD, Danielle PruittiD, Jose BarredaiD, Nathan O. StitzieliD, Paul P. ChengiD, Yongjian LiuiD, , Kory J. LavineiD
Abstract
Vascular smooth muscle cell (VSMC) and immune cell diversification play a central role in driving atherosclerotic coronary artery disease (CAD)1-3. However, the molecular mechanisms governing cell state transitions within the neo-intima in human CAD remain poorly understood, and no lipid-independent therapies are currently approved for its treatment. Here, we performed multi-omic single-cell gene expression profiling, epitope mapping, and spatial transcriptomics from 27 human coronary arteries. Our analysis identified fibroblast activation protein (FAP) as a marker of modulated VSMCs within the neo-intima. Genetic lineage tracing in mice confirmed that FAP cells in the plaque originate from medial VSMCs. Additionally, non-invasive positron emission tomography (PET) imaging in patients with CAD revealed focal FAP uptake in atherosclerotic lesions. Spatial transcriptomics further delineated the distinct localization of VSMC and immune cell subsets within plaques, with FAP states enriched in the neo-intima. To explore the therapeutic potential of targeting de-differentiated VSMCs, we developed an anti-FAP bispecific T-cell engager (BiTE) and demonstrated that it significantly reduced the plaque burden in multiple mouse models of atherosclerosis. Collectively, our study provides the first single-cell and spatially resolved map of human CAD, establishes FAP as a marker of modulated smooth muscle cells, and demonstrates the broader potential of immunotherapeutics for lipid independent targets in atherosclerotic CAD.
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In-depth summary
Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (11 Mar 2025) · CC BY 4.0
Single-cell and spatial maps of human coronary arteries identify FAP as a marker of SMC-derived modulated cells; an anti-FAP BiTE reduced plaque burden in mouse models.
The molecular control of smooth muscle cell state transitions in the human coronary neo-intima is poorly understood, and no lipid-independent therapy is approved for CAD. Earlier plaque nuclei studies lacked cell-level transcriptomic and surface protein data; this work adds CITE-seq and spatial maps and tests an immunotherapy against a modulated SMC marker.
Key results
ShownA CITE-seq atlas of human coronary arteries resolves 14 stromal states. FAP protein is high on the fibromyocyte (FMC) and chondromyocyte (CMC) modulated SMC states, and intimal FAP is confirmed by immunofluorescence.
27 arteries, 150,340 cells, 14 cell types. FMC/CMC show high FAP; immunofluorescence shows strong intimal FAP and none detectable in media. CAD GWAS enrichment is strongest for FMC markers; UK Biobank links FMC/CMC to CAD and ischemic heart disease. · Single-cell multi-omic map of human CAD; FAP enriched on modulated SMCs; FAP associated gene network
ShownFap+ plaque cells in mice derive from medial vascular SMCs, supported by pseudotime inference in human data and two lineage-tracing approaches.
Palantir: FMC/CMC have high pseudotime. Mouse Myh11 lineage cells mapped to human FMC/CMC states. In Myh11 creERT2 tdTomato mice (24 weeks HFD), most plaque Fap+ cells were tdTomato+; quantification n=4/group. · FAP+ cells are derived from medial vascular SMCs; Figure legends
ShownWeekly anti-FAP BiTE lowered aortic arch plaque burden in acute and chronic Apoe-/- atherosclerosis models without changing plasma cholesterol. It also reduced Fap+ cells and raised ACTA2+ fibrous cap area in aortic root plaques.
Acute model: 4 weeks of treatment, n=8-9/group. Chronic model: 16 weeks HFD, treatment from week 12, n=9-10/group. Cholesterol unchanged; en face plaque reduced vs control BiTE (Mann-Whitney). Exact p-values not given in text. · Anti-FAP BiTE treatment reduces atherosclerotic…; Figure legends
SuggestedSpatial NMF programs and MISTy colocalization place FAP+ FMC/CMC states next to inflammatory programs and a lipid-associated macrophage subset (Mac4) at the media-intima boundary. Mac4 resembles oxLDL-treated THP-1 cells.
Visium NMF: MP5 (TGF-β/ECM) beside MP7 (inflammatory). MISTy: Mac4 co-localizes with FMCs; 12 myeloid subsets. Mac4 signature enriched in THP-1 oxLDL states IVMac1 and IVMac3 with a strong mapping score. · Distinct spatial niches in CAD; FAP+ cells and macrophages co-localize in…; CAD intimal macrophages resemble lipid…
SuggestedRe-analysed 68Ga-FAPI-46 PET/CT showed focal tracer uptake at some coronary plaques or stents in patients, a descriptive link between FAP imaging and calcified or stented lesions.
15 patients, 60 coronary arteries; 58% (35/60) had plaques or stents; 40% of these affected vessels showed focal 68Ga-FAPi-46 uptake matching the lesion. Visual assessment only. · FAPI PET/CT in Human Coronary Arteries; FAPi-PET/CT acquisition; FAPI-PET/CT analysis
Methods, data and reproducibility
CITE-seq of human coronary arteries with Visium FFPE and Xenium spatial transcriptomics (tangram deconvolution, NMF meta-programs, MISTy), Palantir pseudotime, mouse lineage tracing, and anti-FAP BiTE treatment in Apoe-/- mice.
- CITE-seq of human coronary arteries from explanted hearts (transplant), 14 cell types · human · n = 27 arteries; 150,340 cells
- Visium FFPE spatial transcriptomics, healthy/early, moderate, and ruptured or obstructed lesions · human · n = 16 samples (4, 8, 4 by lesion group)
- Xenium in situ with custom 100-gene panel plus base panel · human · n = 11 vessels
- 68Ga-FAPI-46 PET/CT re-analysis, patients with acute myocardial infarction or chronic ischemic cardiomyopathy · human · n = 15 patients; 60 coronary arteries
- Published mouse atherosclerosis scRNA-seq with Myh11 and Cdh5 lineage tracing, re-processed and mapped to human map · mouse
- Myh11 creERT2 Rosa26 tdTomato mice with AAV8-D377Y-mPCSK9, 24 weeks HFD, Fap RNAscope · mouse · n = n=4/group
- Anti-FAP vs control BiTE in male Apoe-/- mice, acute and chronic HFD models · mouse · n = 8-9/group acute; 9-10/group chronic
- In vitro THP-1 macrophage/foam cell scRNA-seq (PMA, oxLDL, Dil-oxLDL conditions) · human (THP-1 cell line) · n = 9,397; 9,016; 13,781; 9,298 cells by condition
- Methods & tools
- CellRanger v6.1, dsb, Seurat v4.0 and v5.1.0, Palantir, tangram, GeneNMF multiNMF, MISTy via liana 1.4.0, PROGENy, Harmony v. 0.1.1, 10x Visium FFPE and Xenium, 68Ga-FAPI-46 PET/CT, RNAscope
- Reagents & environment
- CITE-seq: CellRanger v6.1, dsb in R v4, Seurat v4.0. THP-1 scRNA-seq: Cell Ranger v7.1.0, R v.4.2.1, Seurat v.5.1.0, Harmony v. 0.1.1, scDblFinder v.1.2.0. MISTy via liana 1.4.0.
- Reproducibility gaps
- Data are said to be on a GEO super series, but no accession or URL is printed; the code link is a GitHub user page rather than a specific repository.
Limitations (5)
- How FAP+ modulated SMC states are specified remains incompletely defined and likely involves macrophage interactions. · stated by the authors
- Long-term effects of BiTE therapy on vascular remodeling, immune homeostasis and systemic inflammation are unknown; BiTE specificity and toxicity need evaluation, including in humanized systems. · stated by the authors
- The role of FAP-expressing cells in co-existing fibrotic conditions, including cardiac, renal, and hepatic fibrosis, needs further assessment. · stated by the authors
- The PET analysis is a small, visual, descriptive re-analysis of patients with myocardial infarction or ischemic cardiomyopathy. It has no systematic control comparison and no histology linking uptake to modulated SMCs. · noted in this summary
- BiTE efficacy was shown only in male mice, with a mouse-specific FAP binder, and the text gives no exact effect sizes or p-values. Off-target depletion of other FAP+ cells such as fibroblasts was not assessed. · noted in this summary
Who should read:Read this if you work on vascular smooth muscle plasticity, spatial atlases of atherosclerosis, or cell-state-directed immunotherapy and want evidence for FAP as a target.
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