Erik A. L. Biessen

Intraplaque haemorrhage (IPH) is a hallmark of advanced atherosclerosis and a major risk factor for ischemic stroke and myocardial infarction. Current IPH classification focusses on extravascular erythrocyte presence as a proxy of acute bleeding, where iron detection generally indicates older haemorrhages. While often used interchangeably, a comprehensive analysis of transcriptional and metabolic context and impact of iron and erythrocyte deposition on the plaque is still lacking. Here, we investigate iron as a late-stage IPH hallmark in human atherosclerotic plaques. We analysed erythrocyte-rich, iron-rich, and non-IPH regions in human carotid endarterectomy plaques by re-analysing a published transcriptomic dataset of 43 patient samples. In addition, we performed histological and immune phenotyping to define plaque traits associated with iron versus erythrocyte accumulation. Finally, we performed spatial metabolic profiling to functionally define iron-rich regions. Although iron and erythrocyte deposits frequently co-localised, both co-related with different histological traits. While iron- and erythrocyte-rich regions shared transcriptomic features of advanced plaques compared with non-IPH regions, direct comparison showed differences in gene expression profiles. Iron deposition was associated with increased myeloid cell accumulation and a unique spatial metabolic signature distinct from erythrocyte-rich and non-IPH regions. While sharing many characteristics with IPH plaques, the molecular, cellular and metabolic landscape of iron-rich regions is marked by features of plaque remodelling and repair. This makes iron deposition a unique hallmark of late-stage IPH, extending the current erythrocyte-based definition of IPH.

BACKGROUND: Atherosclerotic cardiovascular disease, characterized by an imbalanced lipid metabolism and a dysregulated immune response, is a major cause of death worldwide. The AhR (aryl hydrocarbon receptor) is a ligand-activated transcription factor that is highly expressed in the liver and primarily known for its role in detoxification. However, recent studies suggest that the AhR also plays a key role in immune regulation, indicating that this receptor can influence the development of atherosclerosis. METHODS: Apoe ^−/− and Apoe ^−/− Ahr ^−/− mice were fed a western-type diet for 12 weeks to induce atherosclerosis. Aortic roots were analyzed for size and composition of atherosclerotic plaques. Plasma samples were characterized for inflammation and lipid levels. Liver samples were analyzed for cytokines and lipid accumulation and subjected to RNA sequencing and kinomics. A PheWAS (Phenome-Wide Association Study) was performed to identify associations of genetic AHR variants with atherosclerotic cardiovascular disease and lipid phenotypes in humans. RESULTS: The number of circulating leukocytes was increased in Apoe ^−/− Ahr ^−/− mice compared with Apoe ^−/− controls. Surprisingly, however, mice lacking Ahr showed significantly smaller plaques than Apoe ^−/− mice, which coincided with strongly reduced plasma cholesterol and triglyceride levels. The liver lipid levels showed a similar effect, indicating a key role of the AhR in lipid metabolism. RNA sequencing of the liver revealed that lipid metabolism pathways were particularly impacted in Apoe ^−/− Ahr ^−/− mice. Furthermore, through kinomics, we identified signaling pathways affected by the absence of Ahr . PheWAS analysis revealed significant associations between the AHR variant rs4410790 and lipid traits, including plasma triglycerides, LDL (low-density lipoprotein), and total cholesterol. CONCLUSIONS: Our study demonstrates a remarkable role for AhR in the pathogenesis of atherosclerosis, interfering with both lipid metabolism and inflammatory pathways. Although the underlying mechanisms remain unclear, these results demonstrate a novel and crucial role for AhR in atherosclerotic cardiovascular disease.

BACKGROUND: Atherosclerosis is a leading cause of cardiovascular diseases, and microRNA-26b (miR-26b) has emerged as a significant regulator in its development. This study investigates the role of nonhematopoietic miR-26b in atherosclerosis. METHODS: To study the specific role of nonhematopoietic cell miR-26b in atherosclerosis development, we used a reverse bone marrow transplantation model combined with 12-week Western-type diet feeding. RESULTS: Nonhematopoietic-specific miR-26b deficiency exacerbated atherosclerosis, characterized by larger plaques with increased collagen and necrotic core content. Enhanced VCAM-1 (vascular cell adhesion molecule 1) expression correlated with elevated leukocyte adhesion in ex vivo perfusion studies. Restoration of miR-26b levels in human coronary artery endothelial cells reduced inflammatory responses and leukocyte adhesion. CONCLUSIONS: Our findings highlight that nonhematopoietic miR-26b plays a protective role in atherosclerosis by modulating endothelial cell function, suggesting potential therapeutic applications for miR-26b mimics in cardiovascular disease management.

The importance of neuron-tumor crosstalk has gained increasing attention, yet its influence on the cellular and molecular landscape of colorectal cancer (CRC) remains largely unexplored. Here, we show that colonic innervation shapes the tumor immune microenvironment in a murine model of colitis-associated CRC. Although neuronal density does not affect tumor number, size, or overall burden, transcriptomic profiling of cells isolated from the tumor of hypo-innervated mice revealed extensive differential gene expression, including genes involved in immunoglobulin (Ig) signaling and the cancer-relevant hallmark ‘avoiding immune destruction’. Flow cytometry analysis of leukocyte populations demonstrated a significant reduction of B cells in the cancerous colon of hypo-innervated mice, notably a decrease in germinal center B cells and an altered class-switching profile, characterized by reduced IgA and increased IgD expression. Fluorescence and transmission electron microscopy showed that colonic B cells are primarily localized in the submucosa near neuronal processes containing varicose release sites, suggesting direct neuron-B cell interactions. Functional assays revealed that adrenergic stimulation of B cells promotes their proliferation and maturation, enhances IL-10 secretion, and alters immunoglobulin profiles. Strikingly, the transcriptome of epinephrine-treated B cells closely mirrors that of plasma cells from human CRC tissues, and the transcriptomic signature of these epinephrine-stimulated B cells associates with poorer patient survival. Together, these findings uncover an adrenergic neuron - B cell axis in CRC, providing evidence for direct neuroimmune interactions that affect B cell maturation and may influence tumor progression and therapeutic responses.

Background: Inflammation is a key driver of atherosclerosis, yet the mechanisms sustaining inflammation in human plaques remain poorly understood. This study uses a network-based approach to identify immune gene programs involved in the transition from low- to high-risk (rupture-prone) human atherosclerotic plaques. Methods: Expression data from human carotid artery plaques, both stable (low-risk, n = 16) and unstable (high-risk, n = 27), were analyzed using Weighted Gene Co-expression Network Analysis (WGCNA). Bayesian network inference, operated on the eigengene values from the WGCNA, further extended the WGCNA analysis, and similarity to the signature of T cell subsets was validated in single-cell RNA sequencing data of human plaques, and a loss-of-function study in a mouse model of atherosclerosis. In silico drug repurposing was performed to identify potential therapeutic targets. Results: Our analysis revealed a distinct gene module with a prominent T cell signature, particularly in unstable plaques. Key regulatory factors, RUNX3, IRF7 and in particular PRDM1, were significantly downregulated in plaque T cells from symptomatic versus asymptomatic patients, indicating a protective role. Additionally, as PRDM1 is downstream of IRF7, we opted for PRDM1 as a key target. T cell-specific Prdm1 deficiency in Western-type diet fed Ldlr knockout mice featured accelerated plaque progression. Finally, as PRDM1 targeting drugs are not yet available, we performed in silico drug repurposing, identifying EGFR inhibitors as promising therapeutic candidates. Conclusions: This study highlights a PRDM1-regulated T cell network that distinguishes high-risk from low-risk plaques and demonstrates the regulatory role of T cell PRDM1 in controlling atherosclerosis, positioning this pathway as a promising therapeutic target.

In the published article, there was an error in the Funding statement. The correct funding should be:This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 764474 (CaReSyAn). VJankowski and JJankowski are funded by the German Research Foundation (DFG) through the Transregional Collaborative Research Center (TRR 219; Project ID 322900939), (subproject S-03), (INST 948/4S-1); CRU 5011 project number 445703531, Cost-Action CA 21165, IZKF Multiorgan complexity in Friedreich Ataxia, and Phase Transition in Disease 1-1), ERA-PerMed (ERA-PERMED2022-202-KidneySign).The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.

Introduction: Carbamylation is a non-enzymatic post-translational protein modification common in patients with uremia that causes pro-atherogenic alterations in plasma proteins. It is abundantly present in late-stage atherosclerotic plaques; however, the pathogenic relevance and functional consequences of this accumulation are not known.Methods: Human atherosclerotic plaque tissue samples were stratified by plaques’ stage and kidney function.Results: Immunohistochemistry revealed a significantly higher carbamylated lysine (carb-lys) abundance in latestage hemorrhaged plaques of chronic kidney disease patients compared to early-stage plaques, and a significant negative correlation to glomerular filtration rate for the advanced plaques. While we saw the difference in the total levels of carbamylation between early and advanced plaques, cellular carbamylation signal, studied in a parallel cohort of stable vs unstable plaques, did not differ between plaque stages but significantly correlated to CD68, PLIN2, and LGALS3 signals. Functional effects of carbamylated LDL (carbLDL) uptake on macrophages were studied in vitro on an in-house developed confocal-based microscale multi-assay platform to screen multiple cellular functions and demonstrated similar foam cell formation compared to the uptake of oxidized LDL (oxLDL). However, in contrast to oxLDL, carbLDL did not induce PPARg reporter gene expression, suggesting differential capacity to induce lipogenic pathways. Moreover, unlike oxLDL, carbLDL did not induce apoptosis or ROS production.Discussion: Taken together, our findings demonstrate an accumulation of carbamylated protein during plaque progression in patients with reduced kidney function. This can be, at least partially, explained by uptake of carbLDL particles by the macrophages. CarbLDL uptake, in turn, can induce foam cell formation but seems less cytotoxic than oxLDL.

Hepatocellular carcinoma (HCC) is a major cause of cancer‐related deaths worldwide. Therapeutic options are limited, and therefore new therapeutic targets are needed. Cathepsins, lysosomal proteases, are implicated in various types of cancer. While intracellular cathepsins have various physiological functions, their extracellular secretion can lead to pathological effects. Cathepsin B (CTSB) stays active at neutral pH and contributes to several liver pathologies, including HCC. However, the mechanisms by which extracellular CTSB contributes to HCC remain unclear. Hence, this study aimed to investigate the role of extracellular CTSB in HCC. Cell and spheroid viability of HepG2 and Huh‐7 cells was assessed after treatment with extracellular and intracellular CTSB inhibitors. A chorioallantoic membrane HCC xenograft model was used to study the effect of combined extracellular CTSB inhibitor and chemotherapy treatment on tumour growth, apoptosis, proliferation and angiogenesis. The UK Biobank proteomics data was used to determine the potential role of CTSB in HCC patients. Inhibition of extracellular CTSB significantly decreased the viability of HepG2 and Huh‐7 cells in both monolayers and spheroids compared to intracellular CTSB inhibition. The chorioallantoic membrane model demonstrated that extracellular CTSB inhibition decreased the ratio of proliferation‐apoptosis and, in the presence of paclitaxel, tumour angiogenesis, which resulted in a smaller tumour mass. Furthermore, compared to healthy controls, HCC patients demonstrated higher plasma levels of CTSB which were associated with a higher mortality risk. In conclusion, targeting extracellular CTSB could be a promising therapeutic strategy for HCC, since it decreases angiogenesis and proliferation, while it increases apoptosis.

Background: Metabolism is increasingly recognized as a key regulator of the function and phenotype of the primary cellular constituents of the atherosclerotic vascular wall, including endothelial cells, smooth muscle cells, and inflammatory cells. However, a comprehensive analysis of metabolic changes associated with the transition of plaque from a stable to a hemorrhaged phenotype is lacking. Methods: In this study, we integrated two large mRNA expression and protein abundance datasets (BIKE, n = 126; MaasHPS, n = 43) from human atherosclerotic carotid artery plaque to reconstruct a genome-scale metabolic network (GEM). Next, the GEM findings were linked to metabolomics data from MaasHPS, providing a comprehensive overview of metabolic changes in human plaque. Results: Our study identified significant changes in lipid, cholesterol, and inositol metabolism, along with altered lysosomal lytic activity and increased inflammatory activity, in unstable plaques with intraplaque hemorrhage (IPH+) compared to non-hemorrhaged (IPH−) plaques. Moreover, topological analysis of this network model revealed that the conversion of glutamine to glutamate and their flux between the cytoplasm and mitochondria were notably compromised in hemorrhaged plaques, with a significant reduction in overall glutamate levels in IPH+ plaques. Additionally, reduced glutamate availability was associated with an increased presence of macrophages and a pro-inflammatory phenotype in IPH+ plaques, suggesting an inflammation-prone microenvironment. Conclusions: This study is the first to establish a robust and comprehensive GEM for atherosclerotic plaque, providing a valuable resource for understanding plaque metabolism. The utility of this GEM was illustrated by its ability to reliably predict dysregulation in the cholesterol hydroxylation, inositol metabolism, and the glutamine/glutamate pathway in rupture-prone hemorrhaged plaques, a finding that may pave the way to new diagnostic or therapeutic measures.

The presence of atherosclerotic plaque vessels is a critical factor in plaque destabilization. This may be attributable to the leaky phenotype of these microvessels, although direct proof for this notion is lacking. In this study, we investigated molecular and cellular patterns of stable and hemorrhaged human plaque to identify novel drivers of intraplaque vessel dysfunction. From transcriptome data of a human atherosclerotic lesion cohort, we reconstructed a co-expression network, identifying a gene module strongly and selectively correlated with both plaque microvascular density and inflammation. Spectrin Beta Non-Erythrocytic 1 (sptbn1) was identified as one of the central hubs of this module (along with zeb1 and dock1) and was selected for further study based on its predominant endothelial expression. Silencing of sptbn1 enhanced leukocyte transmigration and vascular permeability in vitro, characterized by an increased number of focal adhesions and reduced junctional VE-cadherin. In vivo, sptbn1 knockdown in zebrafish impaired the development of the caudal vein plexus. Mechanistically, increased substrate stiffness was associated with sptbn1 downregulation in endothelial cells in vitro and in human vessels. Plaque SPTBN1 mRNA and protein expression were found to correlate with an enhanced presence of intraplaque hemorrhage and future cardiovascular disease (CVD) events during follow-up. In conclusion, we identify SPTBN1 as a central hub gene in a gene program correlating with plaque vascularisation. SPTBN1 was regulated by substrate stiffness in vitro while silencing blocked vascular development in vivo, and compromised barrier function in vitro. Together, SPTBN1 is identified as a new potential regulator of the leaky phenotype of atherosclerotic plaque microvessels.