Multiplexed imaging-driven single-cell analysis of abdominal aortic aneurysm according to C-reactive protein deposition

Abstract

Abdominal aortic aneurysm (AAA) is an age-related, life-threatening condition characterized by the expansion of the abdominal aorta. Serum C-reactive protein (CRP) levels are a prognostic marker for AAA, and CRP accelerates tissue injury when deposited in damaged cell membranes in its monomeric form (mCRP). We previously showed that mCRP deposits in eroded atherosclerotic regions are associated with increases in inflammatory cell infiltration and aortic diameter. To investigate the changes in inflammatory-stromal cellular landscape associated with mCRP deposition, we used Co-Detection by Indexing (CODEX) tissue imaging with 31 nucleotide-barcoded antibodies and single-cell-based unsupervised clustering. AAA cases were categorized into High-CRP (n = 6) and Low-CRP (n = 3) groups based on serum levels and immunohistochemistry scores of mCRP. We identified 47 distinct immune and stromal cell types, revealing significant differences in protein expression between groups. In AAA, stromal cells decreased while immune cells increased. High-CRP cases showed increased M1-like and Ki67⁺ proliferating macrophages, and reduced αSMA⁺ cells, whereas Low-CRP cases exhibited intensified fibrosis with CD163⁺Ki67⁺ proliferating M2-like macrophages. Spatial neighborhood enrichment analysis highlighted the close proximity of CD4⁺FOXP3⁺PDL1⁺ T_reg cells to specific clusters: (1) CD57⁺granzyme B⁺ cytotoxic NK cells, (2) CD31⁺HLA-A⁺ endothelial cells, (3) CD45⁺lymphocytes/CD31⁺ endothelial cells, and (4) CD45⁺CD20⁺ B cells in High-CRP cases. Our findings demonstrate the varying distribution of immune cells and vascular wall phenotypes in AAA according to mCRP deposition levels. Targeting inflammation, specifically the immune cells, macrophages, and fibrosis affected by mCRP, may represent a novel approach to halting the pathogenesis of AAA.