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

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 ### Competing Interest Statement The authors have declared no competing interest. * αSMA : α-Smooth Muscle Actin AAA : Abdominal aortic aneurysm CRP : C-reactive protein CODEX : Co-detection by indexing FFPE : Formalin-fixed paraffin-embedded H&E : Hematoxylin and eosin IHC : immunohistochemistry IRB : Institutional Review Board LAG3 : Lymphocyte-activation gene 3 mCRP : Monomeric CRP pCRP : pentameric form of serum CRP ROI : Region of Interest TMA : Tissue microarray t-SNE : t-distributed stochastic neighbor embedding VSMC : Vascular smooth muscle cell