Abstract
Abdominal aortic aneurysm (AAA) is a common cardiovascular disease resulting in high mortality rate due to rupture. Intraluminal thrombus (ILT) is involved in AAA progression via both biomechanically protective and biochemically destructive properties. In this study, we utilized multiplex immunofluorescence and GeoMx Digital Spatial Profiler to explore the following issues: (a) Is ILT associated with the phenotypic switching of vascular smooth muscle cells (VSMCs) in aortic aneurysms? (b) Does ILT enrich macrophage-like VSMCs in aortic aneurysms? (c) What role do macrophage-like VSMCs play in aortic aneurysms? We found that the proportion of CD68 + SMA+ double-positive cells was significantly increased in AAA with thrombus. Differential gene expression, gene set enrichment and gene signature analyses were performed, in which enrichments were mainly related to VSMC phenotypic switching, matrix remodeling and inflammatory response. The cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) was used to quantify the proportions of immune infiltration and the result was that the proportions of naive B cells, M1-type macrophages, and neutrophils were significantly higher in macrophage-like VSMC-rich areas of AAA with ILT. Furthermore, we predicted IL-6 and IL-1β as feature genes which displayed a positive correlation with neutrophil activation. Further, through in vitro assays, we showed that neutrophil extracellular traps (NETs) induce the phenotypic switching of VSMCs through the NF-κB signaling pathway. This study, based on cutting-edge GeoMx Digital Spatial Profiling technology, systematically revealed the cellular and molecular mechanisms underlying the phenotypic switching of VSMCs into macrophage-like cells in AAA associated with ILT. This discovery provides a novel cellular biology perspective for understanding the destructive role of ILT.
Data availability
The data that support the findings of this study are available from the corresponding author (Chao Song; email: chao.song@vip.163.com) upon reasonable request. The spatial transcriptomic datasets are available in the Genome Sequence Archive (GSA) repository under accession number HRA017020.
References
Baman, J. R. & Eskandari, M. K. What Is an Abdominal. Aortic Aneurysm? Jama. 328, 2280 (2022).
Kent, K. C. Clinical practice. Abdominal aortic aneurysms. N. Engl. J. Med. 371, 2101–2108 (2014).
Di Martino, E. et al. Biomechanics of abdominal aortic aneurysm in the presence of endoluminal thrombus: experimental characterisation and structural static computational analysis. Eur. J. Vascular Endovascular Surg. 15, 290–299 (1998).
Haller, S. J. et al. Intraluminal thrombus is associated with early rupture of abdominal aortic aneurysm. J. Vasc. Surg. 67, 1051–1058e1051 (2018).
Boyd, A. J. Intraluminal thrombus: Innocent bystander or factor in abdominal aortic aneurysm pathogenesis? JVS-vascular Sci. 2, 159–169 (2021).
Ma, X., Xia, S., Liu, G. & Song, C. The Detrimental Role of Intraluminal Thrombus Outweighs Protective Advantage in Abdominal Aortic Aneurysm Pathogenesis: The Implications for the Anti-Platelet Therapy. (2022).
Rong, J. X., Shapiro, M., Trogan, E. & Fisher, E. A. Transdifferentiation of mouse aortic smooth muscle cells to a macrophage-like state after cholesterol loading. Proc. Natl. Acad. Sci. U.S.A. 100, 13531–13536 (2003).
Inamoto, S. et al. TGFBR2 mutations alter smooth muscle cell phenotype and predispose to thoracic aortic aneurysms and dissections. Cardiovascular. Res. 88, 520–529 (2010).
Chen, P. Y. et al. Smooth Muscle Cell Reprogramming in Aortic Aneurysms. Cell Stem Cell 26, 542–557.e511 (2020).
Bennett, M. R., Sinha, S. & Owens, G. K. Vascular Smooth Muscle Cells in Atherosclerosis. Circul. Res. 118, 692–702 (2016).
Butoi, E. et al. Cross-talk between macrophages and smooth muscle cells impairs collagen and metalloprotease synthesis and promotes angiogenesis. Biochim. Biophys. Acta. 1863, 1568–1578 (2016).
Ma, X. et al. Hsa_circ_0087352 promotes the inflammatory response of macrophages in abdominal aortic aneurysm by adsorbing hsa-miR-149-5p. Int. Immunopharmacol. 107, 108691 (2022).
Butoi, E. D. et al. Cross talk between smooth muscle cells and monocytes/activated monocytes via CX3CL1/CX3CR1 axis augments expression of pro-atherogenic molecules. Biochim. Biophys. Acta. 1813, 2026–2035 (2011).
Chen, L. et al. Interaction of vascular smooth muscle cells and monocytes by soluble factors synergistically enhances IL-6 and MCP-1 production. Am. J. Physiol. Heart Circ. Physiol. 296, H987–996 (2009).
Li, P. et al. Cross talk between vascular smooth muscle cells and monocytes through interleukin-1خ▓/interleukin-18 signaling promotes vein graft thickening. Arterioscler. Thromb. Vasc. Biol. 34, 2001–2011 (2014).
Zhang, S. et al. The GSA Family in 2025: A Broadened Sharing Platform for Multi-omics and Multimodal Data. Genom. Proteom. Bioinform. 23 (2025).
Kanehisa, M. Toward understanding the origin and evolution of cellular organisms. Protein sci. Publication Protein Soc. 28, 1947–1951 (2019).
Kanehisa, M. & Goto, S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 27–30 (2000).
Kanehisa, M., Furumichi, M., Sato, Y., Matsuura, Y. & Ishiguro-Watanabe, M. KEGG: biological systems database as a model of the real world. Nucleic Acids Res. 53, D672–d677 (2025).
Ma, X. et al. Mechanisms of AGE-induced VSMC phenotypic switching and macrophage modulation in human abdominal aortic aneurysms. Experimental biology and medicine. (Maywood NJ). 250, 10527 (2025).
Xie, S. et al. Daphnetin suppresses experimental abdominal aortic aneurysms in mice via inhibition of aortic mural inflammation. Experimental Ther. Med. 20, 221 (2020).
Hellenthal, F. A. et al. Circulating biomarkers and abdominal aortic aneurysm size. J. Surg. Res. 176, 672–678 (2012).
Lindeman, J. H. et al. Enhanced expression and activation of pro-inflammatory transcription factors distinguish aneurysmal from atherosclerotic aorta: IL-6- and IL-8-dominated inflammatory responses prevail in the human aneurysm. Clin. Sci. (London England: 1979). 114, 687–697 (2008).
Anidjar, S., Dobrin, P. B., Eichorst, M., Graham, G. P. & Chejfec, G. Correlation of inflammatory infiltrate with the enlargement of experimental aortic aneurysms. J. Vasc. Surg. 16, 139–147 (1992).
Ocana, E., Bohأrquez, J. C., Pأrez-Requena, J., Brieva, J. A. & Rodrأguez, C. Characterisation of T and B lymphocytes infiltrating abdominal aortic aneurysms. Atherosclerosis 170, 39–48 (2003).
Tsuruda, T. et al. Adventitial mast cells contribute to pathogenesis in the progression of abdominal aortic aneurysm. Circul. Res. 102, 1368–1377 (2008).
Nie, H., Qiu, J., Wen, S. & Zhou, W. Combining Bioinformatics Techniques to Study the Key Immune-Related Genes in Abdominal Aortic Aneurysm. Front. Genet. 11, 579215 (2020).
Lu, H. et al. Vascular Smooth Muscle Cells in Aortic Aneurysm: From Genetics to Mechanisms. J. Am. Heart Assoc.. 10, e023601 (2021).
Hong, Y. K. et al. Regulation of matrix reloading by tumor endothelial marker 1 protects against abdominal aortic aneurysm. Int. J. Biol. Sci. 20, 3691–3709 (2024).
Andreeva, E. R., Pugach, I. M. & Orekhov, A. N. Subendothelial smooth muscle cells of human aorta express macrophage antigen in situ and in vitro. Atherosclerosis 135, 19–27 (1997).
Cao, G. et al. Deciphering the Intercellular Communication Between Immune Cells and Altered Vascular Smooth Muscle Cell Phenotypes in Aortic Aneurysm From Single-Cell Transcriptome Data. Front. Cardiovasc. Med. 9, 936287 (2022).
Cao, G. et al. How vascular smooth muscle cell phenotype switching contributes to vascular disease. Cell. communication signaling: CCS. 20, 180 (2022).
Cao, G. et al. Single-cell RNA sequencing reveals the vascular smooth muscle cell phenotypic landscape in aortic aneurysm. Cell. Communication Signal. CCS. 21, 113 (2023).
Klopf, J., Brostjan, C., Neumayer, C. & Eilenberg, W. Neutrophils as Regulators and Biomarkers of Cardiovascular Inflammation in the Context of Abdominal Aortic Aneurysms. (2021).
Fontaine, V. et al. Role of leukocyte elastase in preventing cellular re-colonization of the mural thrombus. Am. J. Pathol. 164, 2077–2087 (2004).
Eliason, J. L. et al. Neutrophil depletion inhibits experimental abdominal aortic aneurysm formation. Circulation 112, 232–240 (2005).
Michel, J. B. et al. Novel aspects of the pathogenesis of aneurysms of the abdominal aorta in humans. Cardiovascular. Res. 90, 18–27 (2011).
Fernández-Ruiz, I. Inflammation: NETs are involved in AAA. Nat. reviews Cardiol. 15, 257 (2018).
Wei, M. et al. Inhibition of Peptidyl Arginine Deiminase 4-Dependent Neutrophil Extracellular Trap Formation Reduces Angiotensin II-Induced Abdominal Aortic Aneurysm Rupture in Mice. Front. Cardiovasc. Med. 8, 676612 (2021).
Duftner, C. et al. High prevalence of circulating CD4 + CD28- T-cells in patients with small abdominal aortic aneurysms. Arterioscler. Thromb. Vasc. Biol. 25, 1347–1352 (2005).
Galle, C. et al. Predominance of type 1 CD4 + T cells in human abdominal aortic aneurysm. Clin. Exp. Immunol. 142, 519–527 (2005).
Schأnbeck, U., Sukhova, G. K., Gerdes, N. & Libby, P. T(H)2 predominant immune responses prevail in human abdominal aortic aneurysm. Am. J. Pathol. 161, 499–506 (2002).
Tang, P. C. et al. Transmural inflammation by interferon-gamma-producing T cells correlates with outward vascular remodeling and intimal expansion of ascending thoracic aortic aneurysms. FASEB J. 19, 1528–1530 (2005).
Pagano, M. B. et al. Complement-dependent neutrophil recruitment is critical for the development of elastase-induced abdominal aortic aneurysm. Circulation 119, 1805–1813 (2009).
Ray, A., Basu, S., Williams, C. B., Salzman, N. H. & Dittel, B. N. A novel IL-10-independent regulatory role for B cells in suppressing autoimmunity by maintenance of regulatory T cells via GITR ligand. J. Immunol. (Baltimore, Md: 1950) 188, 3188–3198 (2012).
Zhou, H. F. et al. Antibody directs properdin-dependent activation of the complement alternative pathway in a mouse model of abdominal aortic aneurysm. Proc. Natl. Acad. Sci. U.S.A. 109, E415–422 (2012).
Middleton, R. K. et al. Characterisation of Interleukin-8 and monocyte chemoattractant protein-1 expression within the abdominal aortic aneurysm and their association with mural inflammation. Eur. J. Vascular Endovascular Surg. 37, 46–55 (2009).
Vucevic, D. et al. Inverse production of IL-6 and IL-10 by abdominal aortic aneurysm explant tissues in culture. Cardiovasc. Pathol. 21, 482–489 (2012).
Cai, Q., Lanting, L. & Natarajan, R. Interaction of monocytes with vascular smooth muscle cells regulates monocyte survival and differentiation through distinct pathways. Arterioscler. Thromb. Vasc. Biol. 24, 2263–2270 (2004).
Tai, H. C. et al. Peroxisome Proliferator-Activated Receptor خ Level Contributes to Structural Integrity and Component Production of Elastic Fibers in the Aorta. Hypertension (Dallas, Tex: 1979) 67:1298–1308 (2016).
Hamblin, M. et al. Vascular smooth muscle cell peroxisome proliferator-activated receptor-خ deletion promotes abdominal aortic aneurysms. J. Vasc. Surg. 52, 984–993 (2010).
Miyake, T. et al. Regression of abdominal aortic aneurysms by simultaneous inhibition of nuclear factor kappaB and ets in a rabbit model. Circul. Res. 101, 1175–1184 (2007).
Qin, Z. et al. Angiotensin II-induced TLR4 mediated abdominal aortic aneurysm in apolipoprotein E knockout mice is dependent on STAT3. J. Mol. Cell. Cardiol. 87, 160–170 (2015).
Liu, Q., Shan, P. & Li, H. Gambogic acid prevents angiotensin IIقinduced abdominal aortic aneurysm through inflammatory and oxidative stress dependent targeting the PI3K/Akt/mTOR and NFقخB signaling pathways. Mol. Med. Rep. 19, 1396–1402 (2019).
Ma, X. et al. miR-195 suppresses abdominal aortic aneurysm through the TNF-خ▒/NF-خB and VEGF/PI3K/Akt pathway. Int. J. Mol. Med. 41, 2350–2358 (2018).
Takahara, Y., Tokunou, T. & Ichiki, T. Suppression of Abdominal Aortic Aneurysm Formation in Mice by Teneligliptin, a Dipeptidyl Peptidase-4 Inhibitor. J. Atheroscler. Thromb. 25, 698–708 (2018).
Wang, J. et al. Astragaloside IV Attenuated 3,4-Benzopyrene-Induced Abdominal Aortic Aneurysm by Ameliorating Macrophage-Mediated Inflammation. Front. Pharmacol. 9, 496 (2018).
Wang, Z. et al. Metformin represses the pathophysiology of AAA by suppressing the activation of PI3K/AKT/mTOR/autophagy pathway in ApoE(-/-) mice. Cell. Biosci.. 9, 68 (2019).
Boyle, J. J., Bowyer, D. E., Weissberg, P. L. & Bennett, M. R. Human blood-derived macrophages induce apoptosis in human plaque-derived vascular smooth muscle cells by Fas-ligand/Fas interactions. Arterioscler. Thromb. Vasc. Biol. 21, 1402–1407 (2001).
Koga, J. & Aikawa, M. Crosstalk between macrophages and smooth muscle cells in atherosclerotic vascular diseases. Vascul. Pharmacol. 57, 24–28 (2012).
Stather, P. W. et al. Meta-analysis and meta-regression analysis of biomarkers for abdominal aortic aneurysm. Br. J. Surg. 101, 1358–1372 (2014).
Acknowledgements
We thank Jiaqian Wang from JMDNA Bio-Medical Technology Co., Ltd. (Shanghai) for the support of GeoMx Digital Spatial Profiler study.
Funding
This work was supported by the Young Scientists Fund of the National Natural Science Foundation of China (Grant No. 82100456 and No. 82400164).
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XYM and BWL performed the experiments. XYM analyzed data and prepared figures and draft. CS designed/supervised the study and collected clinical samples. XYM and CS applied for the funding. CS and QSL reviewed the manuscript. All the authors approved the submission and publication of the manuscript.
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Clinical samples were collected with ethical approval from the research ethics committee of Shanghai Changhai Hospital (approval no: CHEC2022-052). Informed consent was obtained from all subjects and/or their legal guardian(s).
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Ma, X., Liang, B., Lu, Q. et al. Molecular and spatial heterogeneity of macrophage like vascular smooth muscle cells in abdominal aortic aneurysms associated with intraluminal thrombus. Sci Rep (2026). https://doi.org/10.1038/s41598-026-43807-y
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DOI: https://doi.org/10.1038/s41598-026-43807-y
