Biology subjects

Melino, G.

Publications and source records attributed to Melino, G..

4 recordsLinked to original sources

bioRxiv2025

Stage-resolved Spatial Multi-omics Reveals Myeloid Niches in Human Atherosclerotic Plaques

BackgroundAtherosclerosis, a leading cause of heart attack and stroke, involves intricate immune cell dynamics within arterial plaques, yet their spatial organization and functional roles remain elusive. MethodsWe combined Visium HD spatial transcriptomics, imaging mass cytometry, and single-cell RNA-seq across fatty-streak, advanced, and restenotic plaques to map myeloid architectures and relate them to lesion geography and extracellular matrix features. Slingshot trajectory analysis resolved macrophages differentiation path. In vitro, we tested microenvironmental and lipid cues separately: fibronectin (FN) exposure during PMA-driven THP-1 differentiation and oxidized LDL (oxLDL)-induced foam cell formation in murine bone-marrow derived macrophages (BMDMs). ResultsOur analysis identified seven macrophage subsets and two neutrophil populations with distinct spatial distribution and functional roles. In early lesions, neutrophils expressing MMP9, MPO, p47phox, TGF-{beta}1 and arachidonate 5-lipoxygenase (ALOX5), aligned with proteolysis, inflammatory processes, and endothelial-mesenchymal transition features. In advanced plaques, macrophage subsets exhibit specialized functions: Ki67+ proliferative macrophages localized near necrotic cores, sustaining local population; SPP1+ macrophages, enriched in lipid handling and tissue remodeling, are prone to apoptosis/ferroptosis, potentially promoting necrotic core expansion; and C3aR+ macrophages form antigen-presenting niches with elevated HLA-DR and CD74, engaging T cells possibly through CXCL12-CXCR4 signaling. Slingshot trajectories indicated progression from C3aR toward SPP1 remodeling states concentrated at fibronectin-rich rims. In vitro, FN increased MMP9 and TIMP1 in THP-1-derived macrophages, consistent with FN imprinting remodeling features characteristic of SPP1 macrophages in situ. Concurrently, oxLDL-treated BMDMs showed enhanced lipid-handling and remodeling modules consistent with the SPP1 program. ConclusionsThese findings define conserved myeloid niches and support a microenvironment-imprinting model that links ECM composition and lipid loading to macrophage state transitions, providing a framework for microenvironment-targeted therapies to stabilize plaques and mitigate cardiovascular risk.

pathology↗

bioRxiv2024

Molecular and spatiotemporal characterization of cells in murine atherosclerotic plaques

ObjectiveSingle-cell technologies have revolutionized our understanding of the phenotypic and transcriptional diversity of aortic leukocytes in atherosclerotic humans and mice. However, enzymatically dissociated tissues lose the spatial context of plaque cells in situ. Here we utilized imaging mass cytometry (IMC) combining with single-cell RNA sequencing (scRNA-seq) to characterize the spatial distribution dynamics, phenotypic transitions, metabolic and functional phenotypes, and the intercellular interaction networks of plaque cells during atherosclerotic progression. Additionally, the dynamic immune landscape of circulating leukocytes associated with atherosclerosis was characterized using cytometry of time of flight (CyTOF). Approach and ResultsA highly multiplexed IMC panel with 33 metal-conjugated antibodies was designed to generate 11 highly multiplexed histology images of aortic root tissues from ApoE-/- mice on high-fat diet at different stage of atherosclerosis. Using histoCAT, we identified 8 principal cell subtypes with distinct phenotypic and geographic dynamics. Furthermore, IMC-defined cell subsets partially corresponded to scRNA-seq-annotated aortic cell subtypes, including 4 macrophage subsets, neutrophils, smooth muscle cells (SMCs) and SMC-derived SEMs (Stem cell, endothelial cell and macrophage-like cell). Activation of inflammatory pathways, increased oxidative phosphorylation and augmented osteoclast differentiation were observed in macrophage populations, SMCs and SEMs from an early stage to advanced stage of atherosclerosis. Notably, cell neighborhood analysis by IMC uncovered multifaceted cell-cell interactions within the plaque, in particular in neutrophil-mediated interactions with smooth muscle cells and macrophages, which were confirmed by ligand-receptor interactions based on scRNA-seq data. Additionally, characterization of the peripheral immune cells by CyTOF revealed an increased ratio of myeloid cells to lymphocytes, and certain neutrophil and monocyte subpopulations also exhibited enhanced lipid metabolism and glycolysis as well as activated inflammatory signaling. ConclusionThis study provides a dynamic spatiotemporal landscape of atherosclerotic lesions and peripheral leukocytes. The new information based on IMC may help understand atherosclerotic pathology and develop novel therapeutic strategies.

pathology↗

bioRxiv2024

Tumour specimen cold ischemia time impacts molecular cancer drug target discovery

Tumour tissue collections are used to uncover pathways associated with disease outcomes that can also serve as targets for cancer treatment, ideally by comparing the molecular properties of cancer tissues to matching normal tissues. The quality of such collections determines the value of the data and information generated from their analyses including expression and modifications of nucleic acids and proteins. These biomolecules are dysregulated upon ischemia and decomposed once the living cells start to decay into inanimate matter. Therefore, ischemia time before final tissue preservation is the most important determinant of the quality of a tissue collection. Here we show the impact of ischemia time on tumour and matching adjacent normal tissue samples for mRNAs in 1,664, proteins in 1,818 and phosphoproteins in 1,800 cases (tumour and matching normal samples) of four solid tumour types (CRC, HCC, LUAD and LUSC NSCLC subtypes). In CRC, ischemia times exceeding 15 minutes impacted 12.5% (mRNA), 25% (protein) and 50% (phosphosites) of differentially expressed molecules in tumour versus normal tissues. This hypoxia- and decay-induced dysregulation increased with longer ischemia times and was observed across tumour types. Interestingly, the proteomics analysis revealed that specimen ischemia time above 15 minutes is mostly associated with a dysregulation of proteins in the immune response pathway and less so with metabolic processes. We conclude that ischemia time is a crucial quality parameter for tissue collections used for target discovery and validation in prognostic cancer research.

cancer biology↗

bioRxiv2023

TAp73 regulates mitochondrial dynamics and multiciliated cell homeostasis through an OPA1 axis

Dysregulated mitochondrial fusion and fission has been implicated in the pathogenesis of numerous diseases. We have identified a novel function of the p53 family protein TAp73 in regulating mitochondrial dynamics. TAp73 regulates the expression of Optic atrophy 1, a protein responsible for controlling mitochondrial fusion, cristae biogenesis and electron transport chain function. Disruption of this axis results in a fragmented mitochondrial network and an impaired capacity for energy production via oxidative phosphorylation. Owing to the role of OPA1 in modulating cytochrome c release, TAp73-/- cells also display an increased sensitivity to apoptotic cell death, e.g., via BH3-mimetics. We also show that the TAp73/OPA1 axis has functional relevance in the upper airway, where TAp73 expression is essential for multiciliated cell differentiation and function. Consistently, ciliated epithelial cells of Trp73-/- (global p73 KO) mice display decreased expression of OPA1 and perturbations of the mitochondrial network, which may drive multiciliated cell loss. In support of this, Trp73 and OPA1 gene expression is decreased in COPD patients, a disease characterised by alterations in mitochondrial dynamics. We therefore highlight a potential mechanism involving the loss of p73 in COPD pathogenesis. This work also adds to the growing body of evidence for growth-promoting roles of TAp73 isoforms.

cell biology↗