This update from the π-HuB Secretariat brings you a notable proteomics advance led by Professor Matthias Mann’s team, titled Proteomics reveals spatial and molecular heterogeneities in advanced atherosclerotic carotid artery plaques, published online on 22 June 2026 in Nature Cardiovascular Research.

Carotid plaque rupture is a major cause of ischemic stroke, yet routine clinical indicators cannot accurately judge plaque rupture risk. Fibrous cap thickness is the core morphological marker for plaque vulnerability: thin-cap plaques (TnC, <200 μm) carry high rupture risk, while thick-cap plaques (TkC, ≥200 μm) remain stable. This work applied spatial proteomics to resolve subregional molecular differences between two plaque phenotypes, and identified PCSK9 as a critical local driver of plaque instability.

Research Strategy

The team established an integrated, closed-loop research framework encompassing multi-layer clinical cohorts, histology-guided spatial proteomics, multi-dimensional bioinformatics, and in vitro mechanistic validation:

1. Multi-layer clinical cohorts: 112 carotid endarterectomy plaque specimens for discovery, plus 20 independent plaques for immunofluorescence validation; a serum cohort of 505 patients including 93 matched plaque-serum paired samples for circulating marker translation.

2. Histology-guided spatial proteomics: Pathologists annotated three distinct anatomical subregions (media, fibrous cap, and necrotic core). Tissue micro-samples were captured via laser microdissection and analyzed using timsTOF DIA-PASEF mass spectrometry, quantifying 4,893 unique proteins.

3. Multi-dimensional analytical workflow: Combined Weighted Gene Co-expression Network Analysis (WGCNA), unsupervised subclustering, and XGBoost-based feature selection for target discovery, followed by primary vascular smooth muscle cell (VSMC) perturbation assays for functional verification.

Core Research Results

1. Conventional clinical indicators show limited predictive utility for plaque stability. Models built solely on routine clinical metadata only reach an AUROC between 0.50 and 0.60, with similarly weak predictive performance replicated in the independent serum cohort. These findings underscore the inadequacy of traditional risk assessment models in identifying high-risk thin-cap lesions.

2. Carotid plaques exhibit pronounced spatial proteomic heterogeneity across distinct anatomical compartments. The necrotic core stands out as the primary region enriched with plaque-destabilizing molecular signals, displaying the greatest proteomic divergence between TnC and TkC groups, whereas the medial layer exhibits minimal molecular shifts. Unsupervised clustering further confirms distinct molecular subtypes within each subregion, demonstrating intra-plaque spatial heterogeneity.

3. Plaque-destabilizing molecular alterations predominate within the necrotic core. A total of 454 differentially expressed proteins (DEPs) are detected in the necrotic core, compared to only 98 in the fibrous cap. Key pathological pathways, including lipid metabolism, chronic immune infiltration and extracellular matrix degradation, are synchronously activated across both regions, jointly weakening the structural integrity of protective plaque caps.

4. PCSK9 ranks as the most statistically relevant protein marking thin-cap status, achieving a single-marker cross-validated AUROC of 0.76 and peaking in high-risk necrotic core subtypes. Followed in vitro experiments using oxidized phospholipid POVPC to mimic necrotic inflammatory microenvironment confirmed oxidative stress drives prominent PCSK9 secretion from VSMCs, and PCSK9 knockdown reverses pro-inflammatory and matrix-degrading cellular phenotypes while restoring LDL receptor abundance.

5. Two distinct biomarker panels were generated for stratified clinical use. A combined 7-protein panel sourced from both fibrous cap and necrotic core tissues achieves an AUROC of 0.86, validated via multiplex immunofluorescence on independent plaque samples for precise pathological assessment. By contrast, an optimized 12-protein serum panel only reaches an AUROC of 0.67, restricted by widespread protein dilution and systemic circulating interference, and is only fit for preliminary population-wide risk screening rather than definitive diagnosis.

Study Summary

Different from bulk proteomics that averages whole-lesion signals, this spatially resolved profiling identifies the necrotic core as the primary molecular hub driving plaque rupture. It elucidates local, hepatic-independent destabilizing functions of VSMC-derived PCSK9 and establishes an end-to-end translational pipeline for carotid plaque risk stratification and primary stroke prevention.

Relevance for the Scientific Mission of π-HuB

This research provides valuable strategic insights for the implementation of the π-HuB initiative: Spatially resolved proteomics proves essential for resolving tissue micro-heterogeneity, establishing a technical precedent for high-resolution human tissue proteome mapping; The end-to-end framework spanning spatial omics discovery to multi-cohort validation offers a reference methodology for biomarker development; the integrated tissue and cellular validation paradigm sets a robust standard for therapeutic target discovery; the divergent performance between tissue- and serum-based markers informs the design of tiered, multi-modal screening frameworks for population health management.

Reference

Sinha A, Sachs N, Kratz E, et al. Proteomics reveals spatial and molecular heterogeneities in advanced atherosclerotic carotid artery plaques. Nat Cardiovasc Res. 2026;5(7):605-623. doi:10.1038/s44161-026-00827-1