Single-Cell Analysis as a Reductionist Approach to Deep Knowledge of Pathogenesis and Pathophysiology of Abdominal Aortic Aneurysm

An abdominal aortic aneurysm (AAA) is a life-threatening condition marked by aortic wall expansion and weakening, with a rupture mortality rate exceeding 80%.^1,2 Various factors contribute to AAA onset and progression, including smooth muscle cell (SMC) apoptosis, elastic fiber fragmentation, extracellular matrix (ECM) degradation, and inflammatory cell infiltration.¹ However, conventional approaches, including bulk RNA sequencing, provide only an averaged tissue view, limiting our understanding of cellular diversity and interactions within AAA lesions. Single-cell RNA sequencing (scRNA-seq) enables comprehensive analysis of gene expression at the individual cell level, and in recent years, multiple scRNA-seq studies of AAA have unveiled previously unknown cellular characteristics (summarized in the Table).^3–9 In 2018, Hadi et al. conducted the first scRNA-seq study on experimental AAA induced by angiotensin II infusion in ApoE knockout mice, identifying macrophage-derived Netrin-1 as a key regulator that promotes matrix metalloproteinase-3 upregulation in vascular SMCs, contributing to extracellular matrix degradation and aneurysm development.³ In 2021, Yang et al. performed scRNA-seq on early-stage (day 4 post-induction) murine AAA induced by perivascular application of CaCl₂, identifying 12 cell clusters across 8 distinct cell types, including SMCs, fibroblasts, macrophages, B/T lymphocytes, neutrophils, and dendritic cells, which were characterized and compared between AAA and sham groups.⁴ In 2021, Davis et al. conducted the first scRNA-seq study on human AAA tissue as compared with non-aneurysmal aortic tissue from patients undergoing open aortobifemoral bypass.⁶ Using this dataset, a 2022 study by the same group provided the first comprehensive catalog of the cellular composition and immune landscape in human AAA.⁸

Table.

Summary of scRNA-seq Studies of AAA

Reference	Model/system	Key findings
Hadi et al. (2018)3	AngII-infused ApoE−/− mice (suprarenal AAA)	First scRNA-seq study on AAA. Macrophage-derived Netrin-1 promoted MMP3 upregulation in VSMCs, contributing to extracellular matrix degradation and aneurysm development
Yang et al. (2021)4	Perivascular CaCl2-induced model in C57BL/6J mice (early-stage infrarenal AAA)	High-resolution single-cell atlas of early-stage AAA. Unbiased scRNA-seq clustering revealed 12 distinct cell populations (across 8 cell types)
Zhao et al. (2021)5	Periadventitial elastase model in C57BL/6J mice (infrarenal AAA)	Characterized cellular heterogeneity during AAA progression and identified 17 clusters (9 lineages) including 4 SMC subpopulations and 5 monocyte/ macrophage subpopulations
Davis et al. (2021)6	Human AAA tissue vs. non-aneurysmal aortic tissue from patients undergoing open aortobifemoral bypass	First scRNA-seq of human AAA found that aneurysmal aorta monocytes/ macrophages highly express JMJD3, a histone demethylase
Qian et al. (2022)7	AngII-infused ApoE−/− mice	ScRNA-seq of AAA vs. control aortas revealed distinct VSMC subpopulations with upregulated α-actinin-2 and mechanosensitive ion channel Piezo1
Davis et al. (2022)8	Reanalysis of Davis et al. (2021)6 scRNA-seq dataset: Human AAA tissue vs. non-aneurysmal aortic tissue	First comprehensive catalog of the cellular composition and immune landscape in human AAA ScRNA-seq identified 17 distinct cell clusters across 8 major cell lineages
Xiao et al. (2024)9	Human AAA tissue	ScRNA-seq of human AAA tissue identified NRF2 as a key regulator of VSMC phenotype switching, promoting the contractile phenotype

AAA, abdominal aortic aneurysm; AngII, angiotensin II; CaCl₂, calcium chloride; ECM, extracellular matrix; scRNA-seq, single-cell RNA sequencing; SMC, smooth muscle cell; VSMC, vascular smooth muscle cell.

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Medial SMCs undergo significant degeneration in AAA, shifting from a contractile to a synthetic/inflammatory phenotype, reducing actin/myosin expression, and weakening the vessel wall.¹⁰ Single-cell analysis has helped visualize this SMC phenotypic switch. In the CaCl₂-induced AAA in mice, Myh11 and Acta2 (which encode smooth muscle myosin heavy chain and actin, respectively) were downregulated, while proliferation- and inflammation-related genes increased.⁴ Strong interactions between SMCs and bone marrow-derived immune cells (e.g., macrophages) were demonstrated.⁸ AAA lesions show upregulated binding of SMC-derived CCL2 (C-C motif chemokine ligand 2) to CCR2 (C-C motif chemokine receptor 2)-positive macrophages and T-cell-derived TNFSF12 (tumor necrosis factor superfamily member 12, also known as TWEAK) to TNFRSF25 (TNF receptor superfamily member 25) receptors on SMCs.⁸

Inflammation is central to AAA progression. Among immune cells, macrophages infiltrate AAA lesions, releasing cytokines and proteases that degrade the vessel wall.¹ Recent single-cell studies have revealed macrophage heterogeneity and the functional stratification of other immune cells in AAA.^4,8 In a single-cell analysis of murine AAA, macrophages clustered into 3 functional subsets: pro-inflammatory, anti-inflammatory, and proliferative. As well as macrophages, diverse immune cells, including T and B lymphocytes and natural killer (NK) cells, play an elaborate and unique role in the pathogenesis/pathophysiology of AAA. Cell–cell interaction analyses highlight pathways through which T-cell-derived factors modulate SMCs and macrophages. Some AAA lesions contain B-cell follicles, potentially modulating local inflammation via antibody and cytokine secretion.⁸ Neutrophils infiltrate early-stage AAA lesions, releasing matrix metalloproteinases and elastases,⁴ while dendritic cells and NK cells are present in smaller numbers, likely contributing to antigen presentation and cytotoxic responses.⁴ Altogether, immune cells in AAA form a complex, chronic inflammatory network that drives tissue remodeling.

Fibroblasts, predominantly located in the arterial adventitia, play key roles in ECM remodeling. ScRNA-seq of murine CaCl₂-induced AAA identified 2 primary fibroblast subclusters: stable and activated.⁴ Notably, the activated fibroblast subset overexpressed cell cycle-related genes such as Mki67, leading to excessive fibrosis. Fibroblast-derived collagenase, elastase-like enzymes, and Tgf-β have been implicated in AAA, supporting their role in ECM degradation and deposition. Interestingly, some AAA fibroblasts co-express smooth muscle markers (e.g., Acta2), suggesting potential fibroblast-to-SMC plasticity. This could indicate that either SMCs transition into fibroblast-like cells or fibroblasts acquire myofibroblast characteristics (e.g., smooth muscle actin positivity). Myofibroblasts help maintain aneurysm wall strength but may also cause pathological stiffening due to excessive ECM deposition. Overall, fibroblasts actively remodel AAA tissue, playing a critical role alongside SMCs and immune cells.

AAA is increasingly recognized as a disorder in which immune responses and inflammation drive disease progression. In this issue of the Journal, Li et al. present their single-cell analysis aimed at further elucidating AAA molecular mechanisms, with a particular focus on lactylation.¹¹ Lactylation, a lactate-driven post-translational modification, adds lactyl groups to lysine residues under high glycolysis conditions. It is implicated in immune regulation, inflammation, and metabolic pathways, with growing interest in its role in both cancer immunity and cardiovascular disease. This current study reveals heterogeneity in lactylation levels across different immune cell types, showing that higher lactylation correlates with increased immune activity. The authors identified 65 lactylation-associated genes, 8 lactylation-related hub genes of which are linked to key immune cell populations and signaling pathways, highlighting the epigenetic-metabolic crosstalk in vascular remodeling and enhancing our understanding of AAA pathogenesis/pathophysiology.

As discussed above, single-cell analysis is a powerful and unbiased method for obtaining a “snapshot” description of gene expression pattern across the various cell types in AAA lesion. Nevertheless, when we interpret this excellent transcriptomic atlas on AAA, there are some issues to be addressed. First, the molecular profiles differ between the experimentally-induced AAA in mice and spontaneous likely-atherosclerotic AAA in humans. Second, the cellular heterogeneity pattern and transcriptomic pattern in AAA might be changing from the initial occurrence phase through the rapid expansion phase (temporal change). Along with the AAA diameter, the morphology and anatomical location (e.g., infrarenal or suprarenal), as well as systemic blood pressure, are related to local wall stress in the AAA lesion. Mechanical and hemodynamic stress to the local AAA wall would considerably influence the transcriptomic pattern in AAA, which could spatially vary according to the location within the AAA lesion (spatial variation). Well-designed spatiotemporal transcriptome analysis is warranted to overcome these issues, and thus, this reductionist approach will successfully help us gain deep knowledge of the pathogenesis/pathophysiology of AAA, paving the way for novel therapeutic targets.

Funding / Conflicts of Interest

None.

Disclosures

H.M. is a member of Circulation Journal’s Editorial Team.

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