Unraveling the Complexity of Abdominal Aortic Aneurysm: Multiplexed Imaging Insights into C-Reactive Protein-Related Variations

ABSTRACT Background Abdominal aortic aneurysm (AAA) is a potentially lethal condition that often remains asymptomatic until it ruptures. Recent research suggests that immune-inflammatory processes are associated with AAA development, yet the exact mechanisms remain unclear. Serum C-reactive protein (CRP) serves as a prognostic marker for AAA and various cardiovascular diseases. When CRP accumulates in damaged tissues, it transforms into a monomeric form, exacerbating tissue damage. Our previous study confirmed the presence of CRP deposition in eroded AAA and atherosclerosis regions, accompanied by an increased infiltration of inflammatory cells. However, a comprehensive understanding of the specific changes in the inflammatory cellular landscape attributable to CRP deposition is lacking. Here, we aimed to explore cellular-level alterations in AAAs associated with varying CRP levels. Methods We categorized AAA patients into High-CRP (≥0.1 mg/dL, and ≥3+ CRP IHC score, n=6) and Low-CRP (≤0.1 mg/dL, and ≤1+ CRP IHC score, n=3), and used normal aorta specimens as a baseline control. The cellular landscape of immune and stromal components was characterized using Co-Detection by Indexing (CODEX) tissue imaging with 31 DNA-barcoded antibodies, followed by single-cell-based analysis and GPU-accelerated unsupervised clustering. Results We identified 51 distinct immune and stromal cell types in the cohort and revealed significant differences in protein expression patterns among the groups. In AAA, stromal cells decreased significantly, while immune cell proportions sharply increased. Lag3+ T cell regulators decreased, leading to an increase in CD3+ T cells. The composition of immune cells within atherosclerotic plaques was associated with the degree of CRP deposition in AAA. The High-CRP group showed increased M1 and Ki67+ proliferating macrophages, while the Low-CRP group exhibited intensified fibrosis with M2 macrophages. Conclusions Our study found significant variations in immune cell distribution within AAA walls based on CRP levels. These findings suggest a potential link between CRP-related immune changes and AAA progression. HIGHLIGHTS By performing CODEX (co-detection by indexing) multiplexed imaging on paraffin-embedded, formalin-fixed archived abdominal aortic aneurysm (AAA) tissue samples with varying CRP levels, we segmented 415,365 cells into 51 distinct clusters. In AAA, the presence of CRP deposition led to variations in the spatial relationships, distances, and enrichment patterns among immune and stromal cells.. In AAA-High CRP, there was an intensified infiltration of M1 macrophages and various immune cells observed in the atherosclerotic plaque. In AAA-Low CRP, severe stromal fibrosis was associated with M2 macrophages. Our results suggest that the deposition of CRP into the atherosclerotic plaque alters the immune-stromal landscape in AAA.