While it is well established that the cellular composition of white adipose tissue (WAT) varies between depots, the functional relevance of this heterogeneity remains unclear. By combining spatial and single-nucleus RNA sequencing, we provide a comprehensive map of subcutaneous and visceral (omental, mesenteric, mesocolic, and epiploic) WAT in both men and women. Our analyses reveal shared features, such as the spatial organization of adipogenesis, alongside depot-specific characteristics, including distinct cell-type enrichments and unique cell-cell communication routes. Epiploic WAT stands out by harboring high proportions of serum amyloid A expressing fat cells (encoded by SAA1/SAA2) and several leukocyte populations. Through mechanistic studies, we demonstrate that adipocyte SAA1/SAA2 expression is induced by inflammatory signals, including lipopolysaccharide, and that SAA1 activates immune responses in adipose-resident myeloid cells. Collectively, our findings suggest that visceral WAT exhibits distinct cytoarchitectural properties, with those located near the colon adapting by developing specialized adipocytes and immune cell populations.

Background: Chronic thromboembolic pulmonary disease (CTEPD) is a long-term complication of pulmonary embolism (PE) and can occur with pulmonary hypertension. Inflammation is implicated but its causal role remains unclear.Objectives: Investigate whether local immune activation modifies haemodynamic, inflammatory, and thromboembolic responses after repetitive PE.Methods: In a 30-day prospective study, six pigs underwent repetitive autologous PE containing cyclic GMP–AMP (cGAMP) to induce local immune activation and were compared with six pigs receiving repetitive PE and tranexamic acid-mediated inhibition of endogenous fibrinolysis (TXA). Animals underwent haemodynamic assessment, blood sampling, computed tomography pulmonary angiography (CTPA), lung tissue cytokine quantification, and histological analysis.Results: Repetitive embolization induced acute transient increases in mean pulmonary arterial pressure in both groups, but no animals developed pulmonary hypertension at follow-up (18±3 mmHg in cGAMP-group and 16±2 mmHg in TXA-group, p=0.20). CTPA demonstrated persistent vascular obstruction in both groups, with comparable obstruction percentages (85 [75–87] % vs. 72 [50–80] %, p=0.29). Systemic leukocyte, neutrophil, and lymphocyte counts remained within porcine reference intervals. groups. Lung tissue CXCL10 levels were higher in the cGAMP-group than in the TXA-group (9120±5027 vs. 3763±1904 pg/g, p=0.035), whereas IL-1β, TNF-α, and IL-8 did not differ. Immunohistochemistry demonstrated CXCL10–positive inflammatory cell infiltration within organised thrombi.Conclusions: In this porcine model, repetitive PE combined with local cGAMP exposure increased lung tissue CXCL10 without inducing pulmonary hypertension over 30 days, suggesting that selective activation of the immune system at clot level alone is insufficient to drive a more advanced chronic thromboembolic phenotype.

Rupture of unstable atherosclerotic plaques is a major cause of mortality. Endothelial-to-mesenchymal transition associates with advanced atherosclerotic plaques and contributes to plaque progression. We examined the role of Twist1, a transcription factor that drives endothelial-to-mesenchymal transition, in plaque progression by inducible deletion from endothelial cells in hypercholesterolemic mice (Twist1ECKO Apo-/-). Single-cell RNA sequencing coupled to endothelial cell-tracking reveals that Twist1 promotes endothelial-to-mesenchymal transition in advanced atherosclerotic plaques. Histological analyses demonstrate that endothelial Twist1 promotes plaque growth and hallmarks of plaque stability (collagen, ACTA2-positive cells) and reduces features of instability (necrosis, macrophage accumulation). Analysis of cultured human aortic endothelial cells shows that TWIST1 contributes to endothelial-to-mesenchymal transition by promoting migration and proliferation through the transcriptional coactivator PELP1. Additionally, TWIST1 promotes endothelial cell proliferation via AEBP1-dependent upregulation of COL4A1. These findings challenge the prevailing view that endothelial-to-mesenchymal transition uniquely destabilizes plaques, by suggesting that TWIST1-driven endothelial-to-mesenchymal transition can promote plaque stability, offering new insights into atherosclerosis pathophysiology and therapeutic potential.

Human white adipose tissue undergoes major remodelling during sustained weight gain that may compromise tissue function and drive cardiometabolic comorbidities. Although weight loss reverses many of these complications, the cellular and molecular adaptations of adipose tissue to different weight loss interventions are poorly understood. Here we show how abdominal subcutaneous adipose tissue (SAT) in men and women with severe obesity adapts to modest lifestyle-induced (8-10%) weight loss followed by substantial bariatric surgery-induced (20-45%) weight loss, using single-nucleus RNA sequencing (snRNA-seq) combined with bulk RNA-seq, and three-dimensional light-sheet fluorescence microscopy. To enable interactive exploration, all snRNA-seq data are available in a browsable format on the Single Cell Portal ( SCP2849 ). Lifestyle-induced weight loss activated proadipogenic gene programmes in progenitor cells, indicating early beneficial effects on SAT. Subsequent surgery-induced weight loss drove profound compositional and transcriptional remodelling of SAT, including increased vascularization and marked reduction of myeloid cell populations. Collectively, our study indicates that following major and sustained weight loss, SAT from individuals with severe obesity has the capacity to return to a state comparable to that observed in lean individuals.

Neural tissues are exceptionally sensitive to oxygen deprivation and rely on a dense network of blood vessels to support their extraordinarily high metabolic demands for oxygen, nutrients and clearance of waste products1-4. In birds, one of the metabolically most demanding neural tissue-the retina-lacks internal blood vessels5,6. This raises the question of how such a metabolically demanding neural tissue can function without blood perfusion. Here we show that, while the photoreceptor outer segments in the outer retina have access to oxygen, the inner bird retina operates under chronic anoxia, supported by anaerobic glycolysis in the retinal neurons. We provide evidence that the pecten oculi-a uniquely vascularized structure in the vitreous humour of birds, the function of which has been debated for centuries5-9-supplies the anoxic inner retina with glucose and removes lactic acid. We suggest that the pecten's metabolic support of the bird retina's anoxia tolerance enabled first the evolution of a thick cell-dense, avascular retina, which secondarily served as an exaptation enabling retinal function during high-altitude migrations.

Uremic toxins lead to high risk of adverse pathophysiological outcomes upon chronic kidney dysfunction. However, the specific toxins and mechanisms that are most pathogenic remain unclear. In this study, we investigated bioactivity of 93 putative uremic toxins systematically with morphological profiling (cell painting). As a result, we identified twenty-four bioactive uremic toxins that significantly induced dose-response changes in cell morphology. These toxins were clustered into four groups based on activity fingerprints, which predict their mechanism of action. The first group is Nrf2-activator related toxins. These toxins were strongly correlated to CDDO compounds, which induce HO-1 by Nrf2 or BACH1 regulation. Label-free proteomics analysis further supports the mechanism of action as Nrf-2 activator and the toxins potentially disrupt the oxidative stress response. The second group and third group exerted non-specific toxicities. Considering the second group only included bile acids, their function as detergents to disrupt cellular membranes may have led to non-specific toxicity. The third group correlated various compounds that are non-related to each other such as antibiotics x-206, salinomycin, narasin, and nigericin. The fourth group contained p-cresol and 4-hydroxyindole that disturb DNA replication. Genotoxicity was observed by cell cycle arrest at S phase and phosphorylation of γH2aX at Ser139. Lastly, adapting activity-based proteomics, we found strong evidence that the group 4 toxins form adduct on reactive cysteines on proteins, which may lead to adverse effects to cells. All in all, we provided unbiased classification of uremic toxins by their bioactivity This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

The transcription factor erythroid 2 (NFE2)-related factor 2 (NRF2) is a key regulator of cellular homeostasis. Recent discoveries have identified agonists of NRF2 as inducers of broad cellular resistance to viral infection including SARS-CoV-2. Nevertheless, it is still unclear to what extent NRF2 itself is an inducer of anti-viral immunity and its downstream antiviral effectors have not been mapped. Here, we first demonstrate through specific genetic activation and silencing that NRF2 restricts SARS-CoV-2 replication. We then used a focused CRISPR-activation screen to map antiviral NRF2-inducible effector genes that restrict replication of SARS-CoV-2, Influenza A virus (IAV), Herpes Simplex virus 1 (HSV1) and Vaccinia virus (VACV). This approach allowed us to identify a range of antiviral effectors each of which restrict members of one or more virus families. Importantly, we identified the NRF2-inducible selective autophagy receptor p62/SQSTM1 as a broadly effective restriction factor across all the tested viruses. Importantly, p62 inhibited SARS-CoV-2 replication in cells treated with the lysosomal inhibitor bafilomycin A1, as well as in cells deficient in the autophagy protein ATG5. Similarly, p62 inhibited replication of HSV1 and IAV independently of ATG5 and ATG16L1 respectively.Thus, NRF2 restricts viral replication through a hitherto underappreciated network of antiviral restriction factors effective across multiple virus families. Importantly, we identify p62 as a broadly acting antiviral effector that restricts viral replication independently of canonical autophagy.

Adipose tissue homeostasis depends on an intact vascular network that ensures adequate nutrient delivery and immune regulation. In obesity, vascular dysfunction, particularly within endothelial cells (ECs), contributes to inflammation and metabolic disease progression, yet the cellular organization of the human adipose vasculature remains poorly defined. Here we show, using single-cell RNA sequencing of nearly 70,000 vascular cells from human subcutaneous adipose tissue of 65 individuals, that the adipose vasculature is highly heterogeneous and consists of seven canonical EC subtypes. In addition, we identify a distinct population of ECs that display mixed endothelial, mesenchymal, adipocytic and immune transcriptional features. Computational analyses and whole-mount imaging support their presence and suggest that they emerge through endothelial-to-mesenchymal transition. Comparative analyses further reveal inflammatory and fibrotic vascular signatures in obesity and type 2 diabetes. Together, this atlas delineates the cellular complexity of the human adipose vasculature and highlights its contribution to metabolic disease.

The endothelium plays a central role in maintaining vascular homeostasis by orchestrating vascular tone, inflammation, healing, permeability, and thrombosis. Assessing endothelial function in vascular tissue is essential for understanding the cellular and molecular mechanisms underlying cardiovascular physiology and pathology. Traditional approaches, such as wire and pressure myography, have been instrumental in defining endothelium-dependent responses and identifying key pharmacological targets. However, the complexity and heterogeneity of endothelial cells across vascular beds and their dynamic phenotypic changes in health and disease necessitate the incorporation of new investigative strategies. Emerging methodologies, including bulk and single-cell transcriptomics, proteomics, and advanced imaging, now provide unprecedented insights into endothelial cell diversity and function. A team of leading experts in the field, who collectively reached a consensus on the most widely used techniques to evaluate endothelial function, developed these guidelines. The document establishes best practices for assessing endothelial function, from endothelial cell cultures to isolated vascular tissues, integrating conventional functional assays with modern molecular approaches. By fostering methodological consistency and embracing innovation, our goal is to enhance rigor, reproducibility, understanding, and discovery in endothelial biology.

Diabetes mellitus (DM) and obesity frequently coexist. Both are associated with adipose dysfunction, yet the contribution of DM remains uncertain. Using bulk transcriptomics of subcutaneous and visceral adipose tissue (SAT and VAT, respectively), we show that DM is associated with shared and distinct patterns of differential gene expression in these depots. Gene ontology analysis of hits across depots highlighted extracellular matrix, inflammatory pathways, metabolism, axon guidance and endoplasmic reticulum stress. Histology revealed larger SAT adipocytes in people with DM, but only in the overweight category. Body mass index (BMI)-stratified transcriptomic analyses of SAT identified DM-associated hits present only in the overweight group. These were validated in plasma protein form using UK Biobank, informing our development of an adipose risk score that predicted incident DM in overweight people beyond a clinical risk score. Hence, molecular signatures of diabetic SAT can define high-risk adiposity, which may aid the targeting of clinical interventions. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by the British Heart Foundation (RG/F/22/110076). DM, MTK and RMC are supported in part by the National Institute for Health and Care Research (NIHR) Leeds Biomedical Research Centre (BRC) (NIHR203331). The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Only existing public datasets were used. Details of the GTEx study procedures can be found at https://gtexportal.org/. UK Biobank received ethical approval from the National Health Service (NHS) Research Ethics Service (11/NW/0382); further details are provided at https://www.ukbiobank.ac.uk/. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data are available upon application to the GTEx portal (https://gtexportal.org/) and to UK Biobank (https://www.ukbiobank.ac.uk/)

Adipose tissue (AT) is a complex connective tissue with a high relative proportion of adipocytes, which are specialized cells with the ability to store lipids in large droplets. AT is found in multiple discrete depots throughout the body, where it serves as the primary repository for excess calories. In addition, AT has an important role in functions as diverse as insulation, immunity and regulation of metabolic homeostasis. The Human Cell Atlas Adipose Bionetwork was established to support the generation of single-cell atlases of human AT as well as the development of unified approaches and consensus for cell annotation. Here, we provide a first roadmap from this bionetwork, including our suggested cell annotations for humans and mice, with the aim of describing the state of the field and providing guidelines for the production, analysis, interpretation and presentation of AT single-cell data. In this Review, the authors present a roadmap towards achieving consensus on development, analysis and interpretation of single-cell transcriptomics data in adipose tissue, including discussion of roadblocks, best practices and ideal cell-type markers for annotation of adipose tissue cell types in mice and humans.

Endothelial cells (ECs) are critical regulators of vascular function and exhibit specialized, organ-specific roles across tissues. During aging, these cells become dysfunctional, resulting in increased susceptibility to cardiovascular disease and its associated mortality. While single-cell transcriptomics studies have revealed extensive endothelial heterogeneity across tissues and conditions, a comprehensive atlas of human EC transcriptomes over the course of the adult human lifespan is still lacking. Here, we present the Human Aging Endothelial Cell Atlas (HAECA), a harmonized single-cell transcriptomic compendium of over 375,000 ECs from 12 human tissues throughout adulthood. Using HAECA, we identified age-associated transcriptional shifts, including a decline in angiogenic gene expression in venous ECs and widespread alterations in extracellular matrix (ECM)- and mechanotransduction-associated pathways. We validated these findings in aging human skin and further uncovered a p21-linked transcriptional program in ECs, confirmed in both in vitro and in vivo models and linked to cellular senescence. Together, our study provides a high-resolution transcriptome reference across spatial as well as temporal axes of the human endothelium. ### Competing Interest Statement The authors have declared no competing interest.

Obesity-driven pathological expansion of white adipose tissue (WAT) is a key driver of endothelial dysfunction. However, early vascular alterations associated with over-nutrition also serve to exacerbate WAT dysfunction. Here, we conduct a single-cell transcriptomic analysis of WAT endothelium to delineate endothelial heterogeneity and elucidate vascular alterations and its consequence in a male murine model of obesity. We demarcate depot-specific differences in subcutaneous (sWAT) and visceral WAT (vWAT) endothelium through in sillico analysis and further corroboration of our findings. Moreover, we identify a sWAT-specific fenestrated endothelial cell (EC) subtype, which declines in obese conditions. Utilizing systemic anti-VEGFA blockade and genetic Vegfa manipulation, we demonstrate that VEGFA is necessary for maintaining fenestration in sWAT. Additionally, we detect this fenestrated EC subtype in male human WAT, which undergoes reduction in individuals with obesity. Collectively, this atlas serves as a valuable tool for future studies to decipher the functional significance of different WAT EC subtypes.

The molecular mechanisms by which lymphatic vessels induce cell contact inhibition are not understood. Here, we identify the cGMP-dependent phosphodiesterase 2A (PDE2A) as a selective regulator of lymphatic but not of blood endothelial contact inhibition. Conditional deletion of Pde2a in mouse embryos reveals severe lymphatic dysplasia, whereas blood vessel architecture remains unaltered. In the absence of PDE2A, human lymphatic endothelial cells fail to induce mature junctions and cell cycle arrest, whereas cGMP levels, but not cAMP levels, are increased. Loss of PDE2A-mediated cGMP hydrolysis leads to the activation of p38 signaling and downregulation of NOTCH signaling. However, DLL4-induced NOTCH activation restores junctional maturation and contact inhibition in PDE2A-deficient human lymphatic endothelial cells. In postnatal mouse mesenteries, PDE2A is specifically enriched in collecting lymphatic valves, and loss of Pde2a results in the formation of abnormal valves. Our data demonstrate that PDE2A selectively finetunes a crosstalk of cGMP, p38, and NOTCH signaling during lymphatic vessel maturation.

A broad range of brain pathologies critically relies on the vasculature, and cerebrovascular disease is a leading cause of death worldwide. However, the cellular and molecular architecture of the human brain vasculature remains incompletely understood1. Here we performed single-cell RNA sequencing analysis of 606,380 freshly isolated endothelial cells, perivascular cells and other tissue-derived cells from 117 samples, from 68 human fetuses and adult patients to construct a molecular atlas of the developing fetal, adult control and diseased human brain vasculature. We identify extensive molecular heterogeneity of the vasculature of healthy fetal and adult human brains and across five vascular-dependent central nervous system (CNS) pathologies, including brain tumours and brain vascular malformations. We identify alteration of arteriovenous differentiation and reactivated fetal as well as conserved dysregulated genes and pathways in the diseased vasculature. Pathological endothelial cells display a loss of CNS-specific properties and reveal an upregulation of MHC class II molecules, indicating atypical features of CNS endothelial cells. Cell-cell interaction analyses predict substantial endothelial-to-perivascular cell ligand-receptor cross-talk, including immune-related and angiogenic pathways, thereby revealing a central role for the endothelium within brain neurovascular unit signalling networks. Our single-cell brain atlas provides insights into the molecular architecture and heterogeneity of the developing, adult/control and diseased human brain vasculature and serves as a powerful reference for future studies.

Abstract The presence of heterogeneity in responses to oncolytic virotherapy poses a barrier to clinical effectiveness, as resistance to this treatment can occur through the inhibition of viral spread within the tumor, potentially leading to treatment failures. Here we show that 4-octyl itaconate (4-OI), a chemical derivative of the Krebs cycle-derived metabolite itaconate, enhances oncolytic virotherapy with VSVΔ51 in various models including human and murine resistant cancer cell lines, three-dimensional (3D) patient-derived colon tumoroids and organotypic brain tumor slices. Furthermore, 4-OI in combination with VSVΔ51 improves therapeutic outcomes in a resistant murine colon tumor model. Mechanistically, we find that 4-OI suppresses antiviral immunity in cancer cells through the modification of cysteine residues in MAVS and IKKβ independently of the NRF2/KEAP1 axis. We propose that the combination of a metabolite-derived drug with an oncolytic virus agent can greatly improve anticancer therapeutic outcomes by direct interference with the type I IFN and NF-κB-mediated antiviral responses.

As hippocampal neurons respond to diverse types of information 1 , a subset assembles into microcircuits representing a memory 2 . Those neurons typically undergo energy-intensive molecular adaptations, occasionally resulting in transient DNA damage 3 – 5 . Here we found discrete clusters of excitatory hippocampal CA1 neurons with persistent double-stranded DNA (dsDNA) breaks, nuclear envelope ruptures and perinuclear release of histone and dsDNA fragments hours after learning. Following these early events, some neurons acquired an inflammatory phenotype involving activation of TLR9 signalling and accumulation of centrosomal DNA damage repair complexes 6 . Neuron-specific knockdown of Tlr9 impaired memory while blunting contextual fear conditioning-induced changes of gene expression in specific clusters of excitatory CA1 neurons. Notably, TLR9 had an essential role in centrosome function, including DNA damage repair, ciliogenesis and build-up of perineuronal nets. We demonstrate a novel cascade of learning-induced molecular events in discrete neuronal clusters undergoing dsDNA damage and TLR9-mediated repair, resulting in their recruitment to memory circuits. With compromised TLR9 function, this fundamental memory mechanism becomes a gateway to genomic instability and cognitive impairments implicated in accelerated senescence, psychiatric disorders and neurodegenerative disorders. Maintaining the integrity of TLR9 inflammatory signalling thus emerges as a promising preventive strategy for neurocognitive deficits.