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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-026-43807-y.