Luke Francis Gamon

Plaque heterogeneity underlies the propensity of atherosclerotic lesions to rupture and trigger cardiovascular events. Most proteomic studies examine bulk changes, obscuring key spatial differences in protein abundance. We report a high-resolution spatial proteomics workflow exploring the molecular landscape of human plaques and a murine myocardium. By combining laser capture microdissection with high-sensitivity ion-mobility mass spectrometry, spatial profiling of cellular and extracellular matrix (ECM) proteomes was achieved. Over 2700 proteins were detected from 50,000 μm2 areas, revealing substantial intraplaque heterogeneity across distinct regions (lipid-rich, media, shoulder, necrotic core, intima) and distance from the artery lumen. Inverse correlations between proteases (cathepsin B) and core structural ECM proteins (perlecan, HSPG2) indicated active ECM remodeling. Analysis of media layers indicated distinct protein signatures associated with smooth muscle contraction and cell–cell communication. Blood coagulation signatures, including platelet degranulation and fibrin formation, were enriched at the intima. Inflammatory (clusters of differentiation 4/68, CD4/CD68; vascular cell adhesion molecule 1, VCAM1) and vascular damage markers (tenascin-C, TNC) were enriched in shoulder regions. The necrotic core was dominated by blood proteins, consistent with intraplaque hemorrhage. This workflow resolves proteomic changes over ∼200 μm distances, providing unprecedented insights into plaque morphology and offers a powerful tool for elucidating plaque biology.

Background and aims: Atherosclerotic plaques form preferentially at vascular sites exposed to disturbed blood flow, yet the protein changes underlying this site-specific plaque development remain unclear. Mouse models are widely used to study atherosclerosis but yield only limited amounts of tissue, previously restricting proteomic studies. However, recent advances in mass spectrometry now enable proteomic profiling of very small tissue samples. We aimed to utilise this to uncover site-specific protein changes in aortic regions prone or resistant to plaque formation. Methods: Aortic arches from apolipoprotein E-deficient (ApoE ^−/− ) mice fed a Western diet (WD) for 16 weeks were dissected into plaques from the major branches and inner curvature and visibly healthy regions. Proteins were extracted, enzymatically digested, and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: More than 4000 proteins were identified per sample despite their small size (< 1 mg tissue). Principal component analysis showed clustering by both disease status and anatomical location within the aortic arch, indicating distinct proteomes. Proteins known to drive atherosclerosis – including vascular cell adhesion molecule 1 (Vcam1), apolipoprotein B (Apob), lipoprotein lipase (Lpl), and galectin 3 (Lgals3) – were most abundant in advanced plaques and decreased progressively across anatomical regions, reaching their lowest levels in ‘healthy’ regions furthest from the plaques. Enrichment analysis highlighted pathways related to the extracellular matrix, immune system, hemostasis, and lipoprotein transport as central to disease progression. Conclusions: This study demonstrates the feasibility of region-resolved proteomics in individual murine aortas and provide new molecular insights into the site-specific nature of atherosclerotic plaque development.

Aims: Vascular atherosclerotic calcification is a pathological process marked by the abnormal deposition of calcium minerals in the intima. Asporin (ASPN) is a small leucine-rich proteoglycan which interacts with collagen and calcium. Due to its role in matrix mineralization, we hypothesized that ASPN might act as a regulator of vascular calcification, thereby promoting atherosclerotic plaque stability. Methods and results: ASPN protein, analysed by ELISA, was quantified in 176 carotid endarterectomy plaques (Carotid Plaque Imaging Project cohort, including 98 patients with cerebrovascular symptoms and 78 asymptomatic patients). Plaque composition was assessed by histological, biochemical, and immunological assays, along with bulk RNA sequencing, to investigate the role of ASPN in atherosclerosis. Patients donating plaques were followed up for post-operative cardiovascular events, median follow-up 6.58 years. The effect of ASPN on smooth muscle cell (SMC) differentiation and matrix mineralization was investigated in vitro using human vascular SMCs overexpressing ASPN. Increased ASPN protein levels were observed in plaques from asymptomatic patients compared with patients with cerebrovascular symptoms. ASPN protein levels were positively associated with markers of plaque stability and regulation of extracellular matrix remodelling while showing an inverse relationship with calcification. Patients with high intraplaque ASPN had a lower risk for future cardiovascular events. Mechanistically, ASPN overexpression in vascular SMCs reduced matrix mineralization in vitro, supporting its potential role in plaque stabilization. Conclusion: ASPN is a regulator of vascular calcification in atherosclerosis, promoting a plaque phenotype that is less prone to rupture. Additionally, high ASPN levels are associated with fewer future cardiovascular events.

The heterogeneity of atherosclerotic plaques is critical for their vulnerability to rupture and the associate risk of cardiovascular events. Most proteomic studies have only examined bulk changes, potentially obscuring key spatial differences in protein content and abundance. Here we report a high-resolution spatial proteomics workflow that allows exploration of the molecular landscape of human plaques and murine myocardial tissue. This combines laser capture microdissection of tissue areas (50,000 µm² from 10 µm-thick sections, corresponding to < 30 cells), with high-sensitivity ion-mobility mass spectrometry, allowing spatial profiling of cellular and extracellular matrix (ECM) proteomes. Over 2700 proteins were detected, revealing substantial intra-plaque proteome heterogeneity across distinct regions (lipid-rich, media layers, shoulder regions, necrotic core, intima) and distance from the lumen into the artery wall. Strong inverse correlations were detected between proteases (e.g. cathepsin-B) and core structural ECM components (e.g. perlecan, HSPG2) consistent with active ECM remodeling. Analysis of media layers indicated distinct protein signatures associated with smooth muscle contraction and cell-cell communication. Blood coagulation signatures, including platelet degranulation and fibrin clot formation were enriched at the intimal surface. Inflammatory markers (clusters of differentiation 4 and 68, CD4/CD68; vascular cell adhesion molecular 1, VCAM1) and vascular damage markers (tenascin-C, TNC) were enriched in shoulder regions. The necrotic core was dominated by blood proteins, consistent with intra-plaque hemorrhage. The capacity of this workflow to resolve changes over modest distances (225 µm) provides unprecedented insights into the spatial organisation of the atherosclerotic microenvironment, offering a powerful tool for elucidating plaque biology and identifying potential therapeutic targets.

At inflammatory sites, immune cells generate oxidants including H₂O₂. Myeloperoxidase (MPO), released by activated leukocytes employs H₂O₂ and halide/pseudohalides to form hypohalous acids that mediate pathogen killing. Hypochlorous acid (HOCl) is a major species formed. Excessive or misplaced HOCl formation damages host tissues with this linked to multiple inflammatory diseases. Previously ( Redox Biology , 2020, 28, 101331) we reported that iodide (I⁻) modulates MPO-mediated protein damage by decreasing HOCl generation with concomitant hypoiodous acid (HOI) formation. HOI may however impact on protein structure, so in this study we examined whether and how HOI, from peroxidase/H₂O₂/I⁻ systems + Cl⁻, modifies proteins. Experiments employed MPO and lactoperoxidase (LPO) and multiple proteins (serum albumins, anastellin), with both chemical (intact protein and peptide mass mapping, LC-MS) and structural (SDS-PAGE) changes assessed. LC-MS analyses revealed dose-dependent iodination of anastellin and albumins by LPO/H ₂ O ₂ with increasing I⁻. Incubation of BSA with MPO/H ₂ O ₂ /Cl⁻ revealed modest chlorination (Tyr286, Tyr475, ∼4%) and Met modification. Lower levels of these species, and extensive iodination at specific Tyr and His residues (>20% modification with > 10 µM I⁻) were detected with increasing I⁻. Anastellin dimerization was inhibited by increasing I⁻, but less marked changes were observed with albumins. These data confirm that I⁻ competes with Cl⁻ for MPO and is an efficient HOCl scavenger. These processes decrease protein chlorination and oxidation, but result in extensive iodination. This is consistent with published data on the presence of iodinated Tyr on neutrophil proteins. The biological implications of protein iodination relative to chlorination require further clarification.

Ex vivo structure determination of macromolecules from native source is gaining increasing attention from the scientific community, as the method can be employed to dissect the function of important, multi-component molecular machines. However, the existing ex vivo procedures often require genome manipulation or availability high-affinity binders, limiting the general applicability. Here, we report simple yet robust principles for isolation of protein complexes from enriched native biological material, enabling cryoEM-facilitated high-resolution structure determination. We report the structures of ten separate membrane and soluble protein complexes determined from spinach leaves. Moreover, the developed pipeline is likely adaptable to essentially any biological system. As such, the approach may represent an attractive avenue for future structural proteomics efforts.