Biology subjects

Davies, M. J.

Publications and source records attributed to Davies, M. J..

5 recordsLinked to original sources

bioRxiv2025

Proteomics and Ex Vivo Plaque Culture Identify the Insulin-Like Growth Factor Axis as a Regulator of Carotid Plaque Stability

ObjectiveRupture of carotid atherosclerotic plaques leading to cerebral embolization, is a significant cause of stroke. We previously analyzed 21 plaques by mass spectrometry and reported that the proteomes of morphologically unstable (rupture-prone) and stable plaques are different. This dataset extends and includes non-atherosclerotic (thyroid) arteries to allow comparison with control tissue, and to investigate plaque stability using ex vivo plaques cultured. MethodsPlaques (n=76) and non-atherosclerotic superior thyroid artery segments (n=8) were retrieved from carotid endarterectomies. Additionally, 22 plaques were cultured ex vivo for 22 days to examine the role of insulin-like growth factor-1 (IGF-1) signalling. Proteins were analyzed by liquid chromatography-mass spectrometry. ResultsMass spectrometric proteome analysis identified three protein clusters associated with mor-phologically unstable (type A) and stable (type B) plaques, as well as non-atherosclerotic arteries. 2,876 proteins were differentially abundant in plaques compared to non-atherosclerotic arteries. 1,415 proteins were differentially abundant between plaque types A and B. Proteins linked to IGF transport and binding, particularly IGF-binding proteins, were more abundant in plaques compared to non-atherosclerotic arteries, and in type B compared to type A plaques. IGF-1, IGF-2 and the IGF-1 receptor were more abundant in type B plaques, whereas the IGF-2 receptor was more abundant in type A. IGF-1 treatment of ex vivo plaques decreased matrix metalloprotein 9 and increased collagen type XXI, consistent with a increased plaque stability. ConclusionsProteomic analyses of atherosclerotic plaques, and ex vivo plaques cultured with IGF-1, re-veals the IGF axis as a potential regulator of human atherosclerotic plaque stability. CLINICAL RELEVANCEThe protein composition of unstable carotid artery plaques differs from that of stable ones, which may explain their varying tendency to rupture. Components of the insulin-like growth factor (IGF) axis are more abundant in atherosclerotic plaques than in healthy tissue, and are more abundant in stable compared to unstable plaque morphology, suggesting a protective role of these proteins against plaque rupture. To investigate this, we treated plaques ex vivo with IGF-1. This data indicates that manipulating the IGF axis may promote plaque stability, with potential clinical relevance in prevention of plaque rupture. ARTICLE HIGHLIGHTSO_ST_ABSType of ResearchC_ST_ABSHuman study Key FindingsProteomic analysis of 76 carotid ath-erosclerotic plaques and 8 superior thyroid artery controls identified the insulin-growth factor (IGF) axis as potential regulators of plaque stability. Ex vivo cul-ture and IGF-1 treatment of 11 symptomatic carotid plaques induced proteome changes, compared to 11 controls, consistent with features of plaque stabiliza-tion. Take home MessageThe insulin-like growth factor axis is a potential regulator of carotid atherosclerotic plaque stability, as revealed by proteomic analysis and ex vivo plaque culture.

molecular biology↗

bioRxiv2025

Spatial proteomics of the human atherosclerotic microenvironment reveals heterogeneity in intra-plaque proteomes and extracellular matrix remodeling

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 {micro}m{superscript 2} from 10 {micro}m-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 {micro}m) provides unprecedented insights into the spatial organisation of the atherosclerotic microenvironment, offering a powerful tool for elucidating plaque biology and identifying potential therapeutic targets.

biochemistry↗

bioRxiv2024

Cleavage of the vascular matrix attracts glioblastoma cells to infiltrate the brain parenchyma

BackgroundGlioblastoma is a highly aggressive brain cancer and, unlike many other cancers types, the median survival for patients after treatment (14.6 months) has barely improved in the last 20 years. Infiltrative growth into the surrounding brain parenchyma facilitates tumor recurrence and ultimately the death of the patient - novel therapies targeting this process are desperately needed. Lysyl oxidase inhibition has been shown to decrease invasive growth in a variety of solid tumours and is a potential therapy for glioblastoma patients. MethodsGenes highly expressed in the mesenchymal subtype of glioblastoma were analyzed in a data set from the Cancer Genome Atlas and tissue microarrays. Two patient-derived human glioblastoma stem cell lines were used to assess the involvement of lysyl oxidase (LOX). The effect of LOX on infiltration was examined in an organotypic brain slice assay and in an orthotopic mouse model. Chemotactic assays, protease and cleavage arrays were used to assess the underlying mechanism behind LOX-mediated infiltration. The orthotopic model was used to evaluate potential clinical utility of targeting LOX in glioblastoma. ResultsLOX is overexpressed in the mesenchymal glioblastoma subtype and strongly associated with poor patient survival. LOX expression upregulates MMP7 expression, which subsequently cleaves the vascular matrix resulting in increased chemotaxis of glioblastoma cells. ConclusionsWe have uncovered a novel mechanism of glioblastoma infiltration and suggest that targeting LOX represent an effective therapeutic approach blocking glioblastoma infiltration. Importance of the studyThe ability of glioblastoma cells to infiltrate the surrounding normal brain tissue facilitates their evasion of current therapies, leading to tumor recurrence and ultimately the death of the patient. To improve targeted therapies for glioblastoma patients we need to understand the molecular mechanisms of glioblastoma cell infiltration and how cells interact with the unique microenvironment of the brain. We have identified a novel mechanism whereby tumor-derived LOX mediates chemotaxis of glioblastoma cells to the laminin rich perivascular niche, enabling infiltrative growth. Inhibiting this infiltrative pathway is a potential anti-invasive therapy that is desperately needed for glioblastoma patients.

cancer biology↗

bioRxiv2024

N-Terminal proteomics reveals distinct protein degradation patterns in different types of human atherosclerotic plaques

BACKGROUNDDestabilization and rupture of atherosclerotic plaques is a major cause of acute atherosclerotic cardiovascular events, including heart attack, ischemic stroke and peripheral arterial disease. Plaque destabilization is associated with extracellular matrix (ECM) modification and remodelling involving protease activity. Enzymatic cleavage generates protein fragments with new ends (N-termini). We hypothesized that plaques susceptible to rupture would contain elevated levels of fragmented proteins with new N-termini. Identification of active proteases and their target proteins might allow categorization of plaque stability. METHODSPlaques from 21 patients who underwent carotid surgery due to symptomatic carotid artery stenosis were examined in an observational/cross-sectional study. The plaques were solubilized, digested, enriched for N-terminal fragments and analyzed by liquid chromatography-mass spectrometry. RESULTSThe above methodology detected 35349 peptides, with 19543 being N-terminal species; 6561 were subsequently identified and quantified. Multidimensional scaling analysis and hierarchical clustering indicate the presence of three distinct clusters, which correlate with gross macroscopic plaque morphology (soft, mixed, and hard), ultrasound classification (echolucent/echogenic) and presence of hemorrhage/ulceration. Major differences were identified in the complement of peptide fragments, consistent with alternative turnover and degradation pathways dependent on plaque type. Identified peptides include signal and pro-peptides from ECM synthesis/turnover, and many from protein fragmentation. Sequence analysis indicates the targeted proteins (including ECM species) and the proteases (including meprins, cathepsins, matrix metalloproteinases, elastase, kallikreins) involved in fragment generation. CONCLUSIONSThis study provides a large dataset of peptide fragments and proteases involved in plaque stability, mechanistic insights into remodelling, and possible biomarkers for improved atherosclerosis risk profiling. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/594251v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@f5e62forg.highwire.dtl.DTLVardef@1db3b07org.highwire.dtl.DTLVardef@7fe3org.highwire.dtl.DTLVardef@cc1329_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Elevated levels of iodide promote peroxidase-mediated protein iodination and inhibit protein chlorination

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/H2O2 with increasing I-. Incubation of BSA with MPO/H2O2/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 {micro}M 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.

biochemistry↗