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In the healthy mature artery, vascular cells, including endothelial cells, smooth muscle cells (SMCs), and fibroblasts are organized in different layers, performing specific functions. SMCs located in the media are in a differentiated state and exhibit a contractile phenotype. However, in response to vascular injury within the intima, stimuli from activated endothelial cells and recruited inflammatory cells reach SMCs and induce a series of remodeling events in them, known as phenotypic switching. Indeed, SMCs retain a certain degree of plasticity and are able to transdifferentiate into other cell types that are crucial for both the formation and development of atherosclerotic lesions. Because of their highly cell-specific expression profiles and their widely recognized contribution to physiological and disease-related biological processes, long noncoding RNAs have received increasing attention in atherosclerosis research. Dynamic fluctuations in their expression have been implicated in the regulation of SMC identity. Sophisticated technologies are now available to allow researchers to access single-cell transcriptomes and study long noncoding RNA function with unprecedented precision. Here, we discuss the state of the art of long noncoding RNAs regulation of SMC phenotypic switching, describing the methodologies used to approach this issue and evaluating the therapeutic perspectives of exploiting long noncoding RNAs as targets in atherosclerosis.

Clonal hematopoiesis (CH) increases inflammasome-linked atherosclerosis but the mechanisms by which CH mutant cells transmit inflammatory signals to non-mutant cells are largely unknown. To address this question we transplanted 1.5% Jak2VF bone marrow (BM) cells with 98.5% WT BM cells into hyperlipidemic Ldlr-/- mice. Low allele burden (LAB) mice showed accelerated atherosclerosis with increased features of plaque instability, decreased levels of macrophage phagocytic receptors MERTK and TREM2, and increased neutrophil extracellular traps (NETs). These changes were reversed when Jak2VF BM was transplanted with Il1r1-/- BM. LAB mice with non-cleavable MERTK in WT BM showed improvements in necrotic core and fibrous cap formation and reduced NETs. An agonistic TREM2 antibody (4D9) markedly increased fibrous caps in both control and LAB mice eliminating the difference between groups. Mechanistically, 4D9 increased TREM2+PDGFB+ macrophages and PDGF receptor-α positive fibroblast-like cells in the cap region. TREM2 and PDGFB mRNA levels were positively correlated in human carotid plaques and co-expressed in macrophages. In summary, low frequency Jak2VF mutations promote atherosclerosis via IL-1 signaling from Jak2VF to WT macrophages and neutrophils promoting cleavage of phagocytic receptors and features of plaque instability. Therapeutic approaches that stabilize MERTK or TREM2 could promote plaque stabilization especially in CH- and inflammasome-driven atherosclerosis.

microRNAs (miRNAs) have been intensively studied as valuable biomarkers in cardiometabolic disease. Typically, miRNAs are detected in plasma or serum, but the use of samples collected in heparinized tubes is problematic for miRNA studies using quantitative PCR (qPCR). Heparin and its derivatives interfere with qPCR-based analysis, leading to a substantial reduction or even complete loss of detectable miRNA levels. Given that red blood cells (RBCs) express abundant miRNAs, whose expression is altered in cardiometabolic disease, RBCs could serve as an attractive alternative in biomarker studies. Here, we aim to explore the stability of miRNAs in RBCs collected from whole blood with different anticoagulants and thereby the potential of RBCs as alternative materials for miRNA biomarker studies. miRNA profiling was performed in human RBCs via RNA sequencing, followed by qPCR validation of selected miRNAs in RBCs and plasma in both heparinized and EDTA tubes. RNA sequencing revealed abundant miRNA presence in RBCs isolated from blood collected in EDTA tubes. miR-210-3p, miR-21-5p, miR-16-5p, and miR-451a were detected at comparable levels in RBCs isolated from both heparinized and EDTA tubes but not in plasma from heparinized tubes. Of note, miR-210-3p levels were consistently lower in RBCs from individuals with type 2 diabetes compared with healthy controls, regardless of anticoagulant type, supporting their potential as biomarker materials. In conclusion, RBCs offer a promising alternative for miRNA biomarker studies, overcoming heparin-related challenges. NEW & NOTEWORTHY microRNAs are valuable biomarkers in cardiometabolic disease, but heparinized tubes hinder their detection because of qPCR interference. RBCs, which express abundant microRNAs like miR-210-3p, may serve as an alternative. microRNAs, including miR-210-3p, are consistently detectable in RBCs at comparable levels between heparinized and EDTA tubes. miR-210-3p levels in RBCs are similarly reduced in heparinized tubes of patients with type 2 diabetes. Thus, RBCs offer a promising solution for miRNA biomarker studies, overcoming heparin-related challenges.

Aims: Although the cannabinoid CB1 receptor has been implicated in atherosclerosis, its cell-specific effects in this disease are not well understood. To address this, we generated a transgenic mouse model to study the role of myeloid CB1 signalling in atherosclerosis., Methods and Results: Here, we report that male mice with myeloid-specific Cnr1 deficiency on atherogenic background developed smaller lesions and necrotic cores than controls, while only minor genotype differences were observed in females. Male Cnr1-deficient mice showed reduced arterial monocyte recruitment and macrophage proliferation with less inflammatory phenotype. The sex-specific differences in proliferation were dependent on oestrogen receptor (ER)α-oestradiol signalling. Kinase activity profiling identified a CB1-dependent regulation of p53 and cyclin-dependent kinases. Transcriptomic profiling further revealed chromatin modifications, mRNA processing, and mitochondrial respiration among the key processes affected by CB1 signalling, which was supported by metabolic flux assays. Chronic administration of the peripherally restricted CB1 antagonist JD5037 inhibited plaque progression and macrophage proliferation, but only in male mice. Finally, CNR1 expression was detectable in human carotid endarterectomy plaques and inversely correlated with proliferation, oxidative metabolism, and inflammatory markers, suggesting a possible implication of CB1-dependent regulation in human pathophysiology., Conclusion: Impaired macrophage CB1 signalling is atheroprotective by limiting their arterial recruitment, proliferation, and inflammatory reprogramming in male mice. The importance of macrophage CB1 signalling appears to be sex-dependent., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Atherosclerosis is an inflammatory disorder responsible for cardiovascular disease. Reactivation of efferocytosis, the phagocytic removal of cells by macrophages, has emerged as a translational target for atherosclerosis. Systemic blockade of the key 'don't-eat-me' molecule, CD47, triggers the engulfment of apoptotic vascular tissue and potently reduces plaque burden. However, it also induces red blood cell clearance, leading to anemia. To overcome this, we previously developed a macrophage-specific nanotherapy loaded with a chemical inhibitor that promotes efferocytosis. Because it was found to be safe and effective in murine studies, we aimed to advance our nanoparticle into a porcine model of atherosclerosis. Here, we demonstrate that production can be scaled without impairing nanoparticle function. At an early stage of disease, we find our nanotherapy reduces apoptotic cell accumulation and inflammation in the atherosclerotic lesion. Notably, this therapy does not induce anemia, highlighting the translational potential of targeted macrophage checkpoint inhibitors., (© 2024. The Author(s).)

The rising incidences of atherosclerosis have necessitated efforts to identify novel targets for therapeutic interventions. In the present study, we observed increased expression of the mechanosensitive calcium channel Piezo1 transcript in mouse and human atherosclerotic plaques, correlating with infiltration of PIEZO1-expressing macrophages. In vitro administration of Yoda1, a specific agonist for PIEZO1, led to increased foam cell apoptosis and enhanced phagocytosis by macrophages. Mechanistically, PIEZO1 activation resulted in intracellular F-actin rearrangement, elevated mitochondrial ROS levels and induction of mitochondrial fragmentation upon PIEZO1 activation, as well as increased expression of anti-inflammatory genes. In vivo, ApoE -/- mice treated with Yoda1 exhibited regression of atherosclerosis, enhanced stability of advanced lesions, reduced plaque size and necrotic core, increased collagen content, and reduced expression levels of inflammatory markers. Our findings propose PIEZO1 as a novel and potential therapeutic target in atherosclerosis., (© 2024. The Author(s).)

Background: Atherosclerosis, a leading cause of cardiovascular disease, involves the pathological activation of various cell types, including immunocytes (eg, macrophages and T cells), smooth muscle cells (SMCs), and endothelial cells. Accumulating evidence suggests that transition of SMCs to other cell types, known as phenotypic switching, plays a central role in atherosclerosis development and complications. However, the characteristics of SMC-derived cells and the underlying mechanisms of SMC transition in disease pathogenesis remain poorly understood. Our objective is to characterize tumor cell-like behaviors of SMC-derived cells in atherosclerosis, with the ultimate goal of developing interventions targeting SMC transition for the prevention and treatment of atherosclerosis., Methods: We used SMC lineage tracing mice and human tissues and applied a range of methods, including molecular, cellular, histological, computational, human genetics, and pharmacological approaches, to investigate the features of SMC-derived cells in atherosclerosis., Results: SMC-derived cells in mouse and human atherosclerosis exhibit multiple tumor cell-like characteristics, including genomic instability, evasion of senescence, hyperproliferation, resistance to cell death, invasiveness, and activation of comprehensive cancer-associated gene regulatory networks. Specific expression of the oncogenic mutant Kras G12D in SMCs accelerates phenotypic switching and exacerbates atherosclerosis. Furthermore, we provide proof of concept that niraparib, an anticancer drug targeting DNA damage repair, attenuates atherosclerosis progression and induces regression of lesions in advanced disease in mouse models., Conclusions: Our findings demonstrate that atherosclerosis is an SMC-driven tumor-like disease, advancing our understanding of its pathogenesis and opening prospects for innovative precision molecular strategies aimed at preventing and treating atherosclerotic cardiovascular disease., Competing Interests: Disclosures None.

The sharing and documentation of cardiovascular research data are essential for efficient use and reuse of data, thereby aiding scientific transparency, accelerating the progress of cardiovascular research and healthcare, and contributing to the reproducibility of research results. However, challenges remain. This position paper, written on behalf of and approved by the German Cardiac Society and German Centre for Cardiovascular Research, summarizes our current understanding of the challenges in cardiovascular research data management (RDM). These challenges include lack of time, awareness, incentives, and funding for implementing effective RDM; lack of standardization in RDM processes; a need to better identify meaningful and actionable data among the increasing volume and complexity of data being acquired; and a lack of understanding of the legal aspects of data sharing. While several tools exist to increase the degree to which data are findable, accessible, interoperable, and reusable (FAIR), more work is needed to lower the threshold for effective RDM not just in cardiovascular research but in all biomedical research, with data sharing and reuse being factored in at every stage of the scientific process. A culture of open science with FAIR research data should be fostered through education and training of early-career and established research professionals. Ultimately, FAIR RDM requires permanent, long-term effort at all levels. If outcomes can be shown to be superior and to promote better (and better value) science, modern RDM will make a positive difference to cardiovascular science and practice. The full position paper is available in the supplementary materials., (© 2023. The Author(s).)

Background: Atherosclerotic plaques are complex tissues composed of a heterogeneous mixture of cells. However, our understanding of the comprehensive transcriptional and phenotypic landscape of the cells within these lesions is limited., Methods: To characterize the landscape of human carotid atherosclerosis in greater detail, we combined cellular indexing of transcriptomes and epitopes by sequencing and single-cell RNA sequencing to classify all cell types within lesions (n=21; 13 symptomatic) to achieve a comprehensive multimodal understanding of the cellular identities of atherosclerosis and their association with clinical pathophysiology., Results: We identified 25 cell populations, each with a unique multiomic signature, including macrophages, T cells, NK (natural killer) cells, mast cells, B cells, plasma cells, neutrophils, dendritic cells, endothelial cells, fibroblasts, and smooth muscle cells (SMCs). Among the macrophages, we identified 2 proinflammatory subsets enriched in IL-1B (interleukin-1B) or C1Q expression, 2 TREM2-positive foam cells (1 expressing inflammatory genes), and subpopulations with a proliferative gene signature and SMC-specific gene signature with fibrotic pathways upregulated. Further characterization revealed various subsets of SMCs and fibroblasts, including SMC-derived foam cells. These foamy SMCs were localized in the deep intima of coronary atherosclerotic lesions. Utilizing cellular indexing of transcriptomes and epitopes by sequencing data, we developed a flow cytometry panel, using cell surface proteins CD29, CD142, and CD90, to isolate SMC-derived cells from lesions. Lastly, we observed reduced proportions of efferocytotic macrophages, classically activated endothelial cells, and contractile and modulated SMC-derived cells, while inflammatory SMCs were enriched in plaques of clinically symptomatic versus asymptomatic patients., Conclusions: Our multimodal atlas of cell populations within atherosclerosis provides novel insights into the diversity, phenotype, location, isolation, and clinical relevance of the unique cellular composition of human carotid atherosclerosis. These findings facilitate both the mapping of cardiovascular disease susceptibility loci to specific cell types and the identification of novel molecular and cellular therapeutic targets for the treatment of the disease., Competing Interests: Disclosures None.

Objective: Skeletal muscle plasticity and remodeling are critical for adapting tissue function to use, disuse, and regeneration. The aim of this study was to identify genes and molecular pathways that regulate the transition from atrophy to compensatory hypertrophy or recovery from injury. Here, we have used a mouse model of hindlimb unloading and reloading, which causes skeletal muscle atrophy, and compensatory regeneration and hypertrophy, respectively., Methods: We analyzed mouse skeletal muscle at the transition from hindlimb unloading to reloading for changes in transcriptome and extracellular fluid proteome. We then used qRT-PCR, immunohistochemistry, and bulk and single-cell RNA sequencing data to determine Mustn1 gene and protein expression, including changes in gene expression in mouse and human skeletal muscle with different challenges such as exercise and muscle injury. We generated Mustn1-deficient genetic mouse models and characterized them in vivo and ex vivo with regard to muscle function and whole-body metabolism. We isolated smooth muscle cells and functionally characterized them, and performed transcriptomics and proteomics analysis of skeletal muscle and aorta of Mustn1-deficient mice., Results: We show that Mustn1 (Musculoskeletal embryonic nuclear protein 1, also known as Mustang) is highly expressed in skeletal muscle during the early stages of hindlimb reloading. Mustn1 expression is transiently elevated in mouse and human skeletal muscle in response to intense exercise, resistance exercise, or injury. We find that Mustn1 expression is highest in smooth muscle-rich tissues, followed by skeletal muscle fibers. Muscle from heterozygous Mustn1-deficient mice exhibit differences in gene expression related to extracellular matrix and cell adhesion, compared to wild-type littermates. Mustn1-deficient mice have normal muscle and aorta function and whole-body glucose metabolism. We show that Mustn1 is secreted from smooth muscle cells, and that it is present in arterioles of the muscle microvasculature and in muscle extracellular fluid, particularly during the hindlimb reloading phase. Proteomics analysis of muscle from Mustn1-deficient mice confirms differences in extracellular matrix composition, and female mice display higher collagen content after chemically induced muscle injury compared to wild-type littermates., Conclusions: We show that, in addition to its previously reported intracellular localization, Mustn1 is a microprotein secreted from smooth muscle cells into the muscle extracellular space. We explore its role in muscle ECM deposition and remodeling in homeostasis and upon muscle injury. The role of Mustn1 in fibrosis and immune infiltration upon muscle injury and dystrophies remains to be investigated, as does its potential for therapeutic interventions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

In this study, organ-on-chip technology is used to develop an in vitro model of medium-to-large size arteries, the artery-on-a-chip (AoC), with the objective to recapitulate the structure of the arterial wall and the relevant hemodynamic forces affecting luminal cells. AoCs exposed either to in vivo-like shear stress values or kept in static conditions are assessed to generate a panel of novel genes modulated by shear stress. Considering the crucial role played by shear stress alterations in carotid arteries affected by atherosclerosis (CAD) and abdominal aortic aneurysms (AAA) disease development/progression, a patient cohort of hemodynamically relevant specimens is utilized, consisting of diseased and non-diseased (internal control) vessel regions from the same patient. Genes activated by shear stress follow the same expression pattern in non-diseased segments of human vessels. Single cell RNA sequencing (scRNA-seq) enables to discriminate the unique cell subpopulations between non-diseased and diseased vessel portions, revealing an enrichment of flow activated genes in structural cells originating from non-diseased specimens. Furthermore, the AoC served as a platform for drug-testing. It reproduced the effects of a therapeutic agent (lenvatinib) previously used in preclinical AAA studies, therefore extending the understanding of its therapeutic effect through a multicellular structure., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

Clonal hematopoiesis (CH) is an independent risk factor for atherosclerotic cardiovascular disease. Murine models of CH suggest a central role of inflammasomes and IL-1β in accelerated atherosclerosis and plaque destabilization. Here we show using single-cell RNA sequencing in human carotid plaques that inflammasome components are enriched in macrophages, while the receptor for IL-1β is enriched in fibroblasts and smooth muscle cells (SMCs). To address the role of inflammatory crosstalk in features of plaque destabilization, we conducted SMC fate mapping in Ldlr -/- mice modeling Jak2 VF or Tet2 CH treated with IL-1β antibodies. Unexpectedly, this treatment minimally affected SMC differentiation, leading instead to a prominent expansion of fibroblast-like cells. Depletion of fibroblasts from mice treated with IL-1β antibody resulted in thinner fibrous caps. Conversely, genetic inactivation of Jak2 VF during plaque regression promoted fibroblast accumulation and fibrous cap thickening. Our studies suggest that suppression of inflammasomes promotes plaque stabilization by recruiting fibroblast-like cells to the fibrous cap., Competing Interests: Competing interests The remaining authors declare no competing interests.

Competing Interests: Disclosures R.A. Boon and S. Dimmeler are listed as inventors on a patent for the use of miR-29 inhibitors to prevent or treat aneurysms (WO2011121120A1). S. Dimmeler has a patent on miR-92. The other author reports no conflicts.

Background: The ADAMTS7 locus was genome-wide significantly associated with coronary artery disease. Lack of the ECM (extracellular matrix) protease ADAMTS-7 (A disintegrin and metalloproteinase-7) was shown to reduce atherosclerotic plaque formation. Here, we sought to identify molecular mechanisms and downstream targets of ADAMTS-7 mediating the risk of atherosclerosis., Methods: Targets of ADAMTS-7 were identified by high-resolution mass spectrometry of atherosclerotic plaques from Apoe -/- and Apoe -/- Adamts7 -/- mice. ECM proteins were identified using solubility profiling. Putative targets were validated using immunofluorescence, in vitro degradation assays, coimmunoprecipitation, and Förster resonance energy transfer-based protein-protein interaction assays. ADAMTS7 expression was measured in fibrous caps of human carotid artery plaques., Results: In humans, ADAMTS7 expression was higher in caps of unstable as compared to stable carotid plaques. Compared to Apoe -/- mice, atherosclerotic aortas of Apoe -/- mice lacking Adamts-7 (Apoe -/- Adamts7 -/- ) contained higher protein levels of Timp-1 (tissue inhibitor of metalloprotease-1). In coimmunoprecipitation experiments, the catalytic domain of ADAMTS-7 bound to TIMP-1, which was degraded in the presence of ADAMTS-7 in vitro. ADAMTS-7 reduced the inhibitory capacity of TIMP-1 at its canonical target MMP-9 (matrix metalloprotease-9). As a downstream mechanism, we investigated collagen content in plaques of Apoe -/- and Apoe -/- Adamts7 -/- mice after a Western diet. Picrosirius red staining of the aortic root revealed less collagen as a readout of higher MMP-9 activity in Apoe -/- as compared to Apoe -/- Adamts7 -/- mice. To facilitate high-throughput screening for ADAMTS-7 inhibitors with the aim of decreasing TIMP-1 degradation, we designed a Förster resonance energy transfer-based assay targeting the ADAMTS-7 catalytic site., Conclusions: ADAMTS-7, which is induced in unstable atherosclerotic plaques, decreases TIMP-1 stability reducing its inhibitory effect on MMP-9, which is known to promote collagen degradation and is likewise associated with coronary artery disease. Disrupting the interaction of ADAMTS-7 and TIMP-1 might be a strategy to increase collagen content and plaque stability for the reduction of atherosclerosis-related events., Competing Interests: Disclosures Dr Schunkert has received personal fees from MSD SHARP & DOHME, AMGEN, Bayer Vital GmbH, Boehringer Ingelheim, Daiichi-Sankyo, Novartis, Servier, Brahms, Bristol-Myers-Squibb, Medtronic, Sanofi Aventis, Synlab, Pfizer, and Vifor T as well as grants and personal fees from Astra-Zeneca outside the submitted work. Drs Schunkert and Kessler are named inventors on a patent application for prevention of restenosis after angioplasty and stent implantation outside the submitted work. Dr Kessler received lecture fees from Bayer, Bristol-Myers Squibb, Abbott, and Astra-Zeneca, which are unrelated to this work. Dr Bernhagen is an inventor on patents related to anti-MIF and antichemokine strategies in inflammation and CVD (cardiovascular diseases) outside the submitted work. The other authors report no conflicts.

Inadequate adaption to mechanical forces, including blood pressure, contributes to development of arterial aneurysms. Recent studies have pointed to a mechanoprotective role of YAP and TAZ in vascular smooth muscle cells (SMCs). Here, we identified reduced expression of YAP1 in human aortic aneurysms. Vascular SMC-specific knockouts (KOs) of YAP/TAZ were thus generated using the integrin α8-Cre (Itga8-Cre) mouse model (i8-YT-KO). i8-YT-KO mice spontaneously developed aneurysms in the abdominal aorta within 2 weeks of KO induction and in smaller arteries at later times. The vascular specificity of Itga8-Cre circumvented gastrointestinal effects. Aortic aneurysms were characterized by elastin disarray, SMC apoptosis, and accumulation of proteoglycans and immune cell populations. RNA sequencing, proteomics, and myography demonstrated decreased contractile differentiation of SMCs and impaired vascular contractility. This associated with partial loss of myocardin expression, reduced blood pressure, and edema. Mediators in the inflammatory cGAS/STING pathway were increased. A sizeable increase in SOX9, along with several direct target genes, including aggrecan (Acan), contributed to proteoglycan accumulation. This was the earliest detectable change, occurring 3 days after KO induction and before the proinflammatory transition. In conclusion, Itga8-Cre deletion of YAP and TAZ represents a rapid and spontaneous aneurysm model that recapitulates features of human abdominal aortic aneurysms.

An abdominal aortic aneurysm (AAA) is a pathological widening of the aortic wall characterized by loss of smooth muscle cells (SMCs), extracellular matrix degradation, and local inflammation. This condition is often asymptomatic until rupture occurs, leading to high morbidity and mortality rates. Diagnosis is mostly accidental and the only currently available treatment option remains surgical intervention. Circular RNAs (circRNAs) represent a novel class of regulatory non-coding RNAs that originate from backsplicing. Their highly stable loop structure, combined with a remarkable enrichment in body fluids, make circRNAs promising disease biomarkers. We investigated the contribution of circRNAs to AAA pathogenesis and their potential application to improve AAA diagnostics. Gene expression analysis revealed the presence of deregulated circular transcripts stemming from AAA-relevant gene loci . Among these, the circRNA to the Ataxia Telangiectasia Mutated gene (c ATM ) was upregulated in human AAA specimens, in AAA-derived SMCs, and serum samples collected from aneurysm patients. In primary aortic SMCs, c ATM increased upon angiotensin II and doxorubicin stimulation, while its silencing triggered apoptosis. Higher c ATM levels made AAA-derived SMCs less vulnerable to oxidative stress, compared with control SMCs. These data suggest that c ATM contributes to elicit an adaptive oxidative-stress response in SMCs and provides a reliable AAA disease signature., Competing Interests: L.M. is a scientific consultant and adviser for Novo Nordisk (Malov, Denmark), DrugFarm (Shanghai, China), and Angiolutions (Hannover, Germany), and received research funds from Roche Diagnostics (Rotkreuz, Switzerland)., (© 2023 The Authors.)

Background: Activation of vascular smooth muscle cell (VSMC) inflammation is vital to initiate vascular disease. The role of human-specific long noncoding RNAs in VSMC inflammation is poorly understood., Methods: Bulk RNA sequencing in differentiated human VSMCs revealed a novel human-specific long noncoding RNA called inflammatory MKL1 (megakaryoblastic leukemia 1) interacting long noncoding RNA ( INKILN ). INKILN expression was assessed in multiple in vitro and ex vivo models of VSMC phenotypic modulation as well as human atherosclerosis and abdominal aortic aneurysm. The transcriptional regulation of INKILN was verified through luciferase reporter and chromatin immunoprecipitation assays. Loss-of-function and gain-of-function studies and multiple RNA-protein and protein-protein interaction assays were used to uncover a mechanistic role of INKILN in the VSMC proinflammatory gene program. Bacterial artificial chromosome transgenic mice were used to study INKILN expression and function in ligation injury-induced neointimal formation., Results: INKILN expression is downregulated in contractile VSMCs and induced in human atherosclerosis and abdominal aortic aneurysm. INKILN is transcriptionally activated by the p65 pathway, partially through a predicted NF-κB (nuclear factor kappa B) site within its proximal promoter. INKILN activates proinflammatory gene expression in cultured human VSMCs and ex vivo cultured vessels. INKILN physically interacts with and stabilizes MKL1, a key activator of VSMC inflammation through the p65/NF-κB pathway. INKILN depletion blocks interleukin-1β-induced nuclear localization of both p65 and MKL1. Knockdown of INKILN abolishes the physical interaction between p65 and MKL1 and the luciferase activity of an NF-κB reporter. Furthermore, INKILN knockdown enhances MKL1 ubiquitination through reduced physical interaction with the deubiquitinating enzyme USP10 (ubiquitin-specific peptidase 10). INKILN is induced in injured carotid arteries and exacerbates ligation injury-induced neointimal formation in bacterial artificial chromosome transgenic mice., Conclusions: These findings elucidate an important pathway of VSMC inflammation involving an INKILN /MKL1/USP10 regulatory axis. Human bacterial artificial chromosome transgenic mice offer a novel and physiologically relevant approach for investigating human-specific long noncoding RNAs under vascular disease conditions., Competing Interests: Disclosures None.

Popliteal artery aneurysm (PAA) is the most frequent peripheral aneurysm, primarily seen in male smokers with a prevalence below 1%. This exploratory study aims to shed light on cellular mechanisms involved in PAA progression. Sixteen human PAA and eight non-aneurysmatic popliteal artery samples, partially from the same patients, were analyzed by immunohistochemistry, fluorescence imaging, Affymetrix mRNA expression profiling, qPCR and OLink proteomics, and compared to atherosclerotic ( n = 6) and abdominal aortic aneurysm (AAA) tissue ( n = 19). Additionally, primary cell culture of PAA-derived vascular smooth muscle cells (VSMC) was established for modulation and growth analysis. Compared to non-aneurysmatic popliteal arteries, VSMCs lose the contractile phenotype and the cell proliferation rate increases significantly in PAA. Array analysis identified APOE higher expressed in PAA samples, co-localizing with VSMCs. APOE stimulation of primary human PAA VSMCs significantly reduced cell proliferation. Accordingly, contractile VSMC markers were significantly upregulated. A single case of osseous mechanically induced PAA with a non-diseased VSMC profile emphasizes these findings. Carefully concluded, PAA pathogenesis shows similar features to AAA, yet the mechanisms involved might differ. APOE is specifically higher expressed in PAA tissue and could be involved in VSMC phenotype rescue.

Competing Interests: Conflict of interest Lars Maegdefessel is a scientific and medical consultant for Novo Nordisk, DrugFarm, and Angiolutions. He and his lab received financial research support from Novo Nordisk and Roche Diagnostics.

Long non-coding RNAs (lncRNAs) orchestrate various biological processes and regulate the development of cardiovascular diseases. Their potential therapeutic benefit to tackle disease progression has recently been extensively explored. Our study investigates the role of lncRNA Nudix Hydrolase 6 (NUDT6) and its antisense target fibroblast growth factor 2 (FGF2) in two vascular pathologies: abdominal aortic aneurysms (AAA) and carotid artery disease. Using tissue samples from both diseases, we detected a substantial increase of NUDT6, whereas FGF2 was downregulated. Targeting Nudt6 in vivo with antisense oligonucleotides in three murine and one porcine animal model of carotid artery disease and AAA limited disease progression. Restoration of FGF2 upon Nudt6 knockdown improved vessel wall morphology and fibrous cap stability. Overexpression of NUDT6 in vitro impaired smooth muscle cell (SMC) migration, while limiting their proliferation and augmenting apoptosis. By employing RNA pulldown followed by mass spectrometry as well as RNA immunoprecipitation, we identified Cysteine and Glycine Rich Protein 1 (CSRP1) as another direct NUDT6 interaction partner, regulating cell motility and SMC differentiation. Overall, the present study identifies NUDT6 as a well-conserved antisense transcript of FGF2. NUDT6 silencing triggers SMC survival and migration and could serve as a novel RNA-based therapeutic strategy in vascular diseases., Competing Interests: Declaration of interests L.M. is a scientific consultant and adviser for Novo Nordisk (Malov, Denmark), DrugFarm (Shanghai, China), and Angiolutions (Hannover, Germany), and received research funds from Roche Diagnostics (Rotkreuz, Switzerland) and Novo Nordisk (Malov, Denmark)., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Despite extraordinary advances in the comprehension of the pathophysiology of atherosclerosis and the employment of very effective treatments, cardiovascular diseases are still a major cause of mortality and represent a large share of health expenditure worldwide. Atherosclerosis is a disease affecting the medium and large arteries, which consists of a progressive accumulation of fatty substances, cellular waste products and fibrous elements, which culminates in the buildup of a plaque obstructing the blood flow. Endothelial dysfunction represents an early pathological event, favoring immune cells recruitment and triggering local inflammation. The release of inflammatory cytokines and other signaling molecules stimulates phenotypic modifications in the underlying vascular smooth muscle cells, which, in physiological conditions, are responsible for the maintenance of vessels architecture while regulating vascular tone. Vascular smooth muscle cells are highly plastic and may respond to disease stimuli by de-differentiating and losing their contractility, while increasing their synthetic, proliferative, and migratory capacity. This phenotypic switching is considered a pathological hallmark of atherogenesis and is ruled by the activation of selective gene programs. The advent of genomics and the improvement of sequencing technologies deepened our knowledge of the complex gene expression regulatory networks mediated by non-coding RNAs, and favored the rise of innovative therapeutic approaches targeting the non-coding transcriptome. In the context of atherosclerosis, long non-coding RNAs have received increasing attention as potential translational targets, due to their contribution to the molecular dynamics modulating the expression of vascular smooth muscle cells contractile/synthetic gene programs. In this review, we will focus on the most well-characterized long non-coding RNAs contributing to atherosclerosis by controlling expression of the contractile apparatus and genes activated in perturbed vascular smooth muscle cells., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Calcification, a key feature of advanced human atherosclerosis, is positively associated with vascular disease burden and adverse events. We showed that macrocalcification can be a stabilizing factor for carotid plaque molecular biology, due to inverse association with immune processes. Mast cells (MCs) are important contributors to plaque instability, but their relationship with macrocalcification is unexplored. With a hypothesis that MC activation negatively associates with carotid plaque macrocalcification, we aimed to investigate the link between MCs and carotid plaque vulnerability, and study MC role in plaque calcification via smooth muscle cells (SMCs)., Methods: Pre-operative computed tomography angiographies of patients (n = 40) undergoing surgery for carotid stenosis were used to characterize plaque morphology. Plaque microarrays (n = 40 and n = 126) were used for bioinformatic deconvolution of immune cell populations. Tissue microarrays (n = 103) were used to histologically validate the contribution of activated and resting MCs in plaques., Results: Activated MCs and their typical markers were negatively correlated with macrocalcification. The ratio of activated vs. resting MCs was increased in low-calcified plaques from symptomatic patients. There was no modulating effect of medication on MC ratios. In vitro experiments showed that SMC calcification attenuated MC activation, while both active and resting MCs stimulated SMC calcification and induced dedifferentiation towards a pro-inflammatory-, osteochondrocyte-like phenotype, without modulating their migro-proliferative function., Conclusions: Integrative analyses from human plaques showed that MC activation is inversely associated with macrocalcification and positively with parameters of plaque vulnerability. Mechanistically, MCs induce SMC osteogenic reprograming, while matrix calcification in turn attenuates MC activation, offering new therapeutic avenues for exploration., Competing Interests: Declaration of Competing Interest The funding bodies and companies were not involved in the study design, manuscript writing or any other involvement in the creation of this manuscript., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Atherosclerosis and numerous other cardiovascular diseases develop in an age-dependent manner. The endothelial cells that line the vessel walls play an important role in the development of atherosclerosis. Non-coding RNA like microRNAs and long non-coding RNAs are known to play an important role in endothelial function and are implicated in the disease progression. Here, we summarize several microRNAs and long non-coding RNAs that are known to have an altered expression with endothelial aging and discuss their role in endothelial cell function and senescence. These processes contribute to aging-induced atherosclerosis development and by targeting the non-coding RNAs controlling endothelial cell function and senescence, atherosclerosis can potentially be attenuated., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: LM is a scientific consultant and adviser for Novo Nordisk (Malov, Denmark), DrugFarm (Shanghai, China), and Angiolutions (Hannover, Germany), and received research funds from Roche Diagnostics (Rotkreuz, Switzerland)., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Aims: Abdominal aortic aneurysm (AAA) is a common cardiovascular disease with a strong correlation to smoking, although underlying mechanisms have been minimally explored. Electronic cigarettes (e-cigs) have gained recent broad popularity and can deliver nicotine at comparable levels to tobacco cigarettes, but effects on AAA development are unknown., Methods and Results: We evaluated the impact of daily e-cig vaping with nicotine on AAA using two complementary murine models and found that exposure enhanced aneurysm development in both models and genders. E-cigs induced changes in key mediators of AAA development including cytokine chitinase-3-like protein 1 (CHI3L1/Chil1) and its targeting microRNA-24 (miR-24). We show that nicotine triggers inflammatory signalling and reactive oxygen species while modulating miR-24 and CHI3L1/Chil1 in vitro and that Chil1 is crucial to e-cig-augmented aneurysm formation using a knockout model., Conclusions: In conclusion our work shows increased aneurysm formation along with augmented vascular inflammation in response to e-cig exposure with nicotine. Further, we identify Chil1 as a key mediator in this context. Our data raise concerns regarding the potentially harmful long-term effects of e-cig nicotine vaping., Competing Interests: Conflict of interest: None declared., (Published by Oxford University Press on behalf of the European Society of Cardiology 2022.)

Background and Aim:  The ability to recognize and monitor atherosclerotic lesion development using noninvasive imaging is crucial in preventive cardiology. The aim of the present study was to establish a protocol for longitudinal monitoring of plaque lipid, collagen, and macrophage burden as well as of endothelial permeability., Methods and Results:  Photoacoustic signals derived from endogenous or exogenous dyes assessed in vivo, in plaques of albino Apoe -/- mice, correlated with lesion characteristics obtained after histomorphometric and immunofluorescence analyses, thus supporting the validity of our protocol. Using models of atheroprogression and regression, we could apply our imaging protocol to the longitudinal observation of atherosclerotic lesion characteristics in mice., Conclusions:  The present study shows an innovative approach to assess arterial inflammation in a non-invasive fashion, applicable to longitudinal analyses of changes of atherosclerotic lesion composition. Such approach could prove important in the preclinical testing of therapeutic interventions in mice carrying pre-established lesions., Competing Interests: None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Background and Aims: Atherosclerosis is a systemic and chronic inflammatory disease propagated by monocytes and macrophages. Yet, our knowledge on how transcriptome of these cells evolves in time and space is limited. We aimed at characterizing gene expression changes in site-specific macrophages and in circulating monocytes during the course of atherosclerosis., Methods: We utilized apolipoprotein E-deficient mice undergoing one- and six-month high cholesterol diet to model early and advanced atherosclerosis. Aortic macrophages, peritoneal macrophages, and circulating monocytes from each mouse were subjected to bulk RNA-sequencing (RNA-seq). We constructed a comparative directory that profiles lesion- and disease stage-specific transcriptomic regulation of the three cell types in atherosclerosis. Lastly, the regulation of one gene, Gpnmb, whose expression positively correlated with atheroma growth, was validated using single-cell RNA-seq (scRNA-seq) of atheroma plaque from murine and human., Results: The convergence of gene regulation between the three investigated cell types was surprisingly low. Overall 3245 differentially expressed genes were involved in the biological modulation of aortic macrophages, among which less than 1% were commonly regulated by the remote monocytes/macrophages. Aortic macrophages regulated gene expression most actively during atheroma initiation. Through complementary interrogation of murine and human scRNA-seq datasets, we showcased the practicality of our directory, using the selected gene, Gpnmb, whose expression in aortic macrophages, and a subset of foamy macrophages in particular, strongly correlated with disease advancement during atherosclerosis initiation and progression., Conclusions: Our study provides a unique toolset to explore gene regulation of macrophage-related biological processes in and outside the atheromatous plaque at early and advanced disease stages., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Abdominal aortic aneurysm (AAA) rupture is still associated with a mortality rate of 80-90%. Imaging techniques or molecular fingerprinting for patient-specific risk stratification to identify pending rupture are still lacking. The chemokine (C-X-C motif) receptor (CXCR4) activation by CXCL12 ligand has been identified as a marker of inflammation and atherosclerosis, associated with AAA. Both are highly expressed in the aortic aneurysm wall. However, it is still unclear whether different expression levels of CXCR4 and CXCL12 can distinguish ruptured AAAs (rAAA) from intact AAAs (iAAA). Patients and methods: Abdominal aortic tissue samples (rAAA: n=29; iAAA: n=54) were excised during open aortic repair. Corresponding serum samples from these patients (n=9 from rAAAs; n=47 from iAAA) were drawn pre-surgery. Healthy aortic tissue samples (n=8) obtained from adult kidney donors during transplantation and serum samples from healthy adult volunteers were used as controls (n=5 each). Results: CXCR4 was mainly expressed in the media of the aneurysmatic tissue. Focal positive staining was also observed in areas of inflammatory infiltrates within the adventitia. In tissue lysates, no significant differences between iAAA, rAAA, and healthy controls were observed upon ELISA analysis. In serum samples, the level of CXCR4 was significantly increased in rAAA by 4-fold compared to healthy controls ( p =0.011) and 3.0-fold for rAAA compared to iAAA ( p <0.001). Furthermore a significant positive correlation between aortic diameter and serum CXCR4 concentration was found for both, iAAA and rAAA ( p =0.042). Univariate logistic regression analysis showed that increased CXCR4 serum concentrations were associated with AAA rupture (OR: 4.28, 95% CI: 1.95-12.1, p =0.001). Conclusions: CXCR4 concentration was significantly increased in serum of rAAA patients and showed a significant correlation with an increased aortic diameter. The level of CXCR4 in serum was associated with a more than 4-fold risk increase for rAAA and thus could possibly serve as a biomarker in the future. However, further validation in larger studies is required.

Background: Long noncoding RNAs (lncRNAs) have emerged as novel regulators of macrophage biology and inflammatory cardiovascular diseases. However, studies focused on lncRNAs in human macrophage subtypes, particularly human lncRNAs that are not conserved in rodents, are limited., Methods: Through RNA-sequencing of human monocyte-derived macrophages, we identified suppressor of inflammatory macrophage apoptosis lncRNA ( SIMALR ). Lipopolysaccharide/IFNγ (interferon γ) stimulated human macrophages were treated with SIMALR antisense oligonucleotides and subjected to RNA-sequencing to investigate the function of SIMALR . Western blots, luciferase assay, and RNA immunoprecipitation were performed to validate function and potential mechanism of SIMALR. RNAscope was performed to identify SIMALR expression in human carotid atherosclerotic plaques., Results: RNA-sequencing of human monocyte-derived macrophages identified SIMALR , a human macrophage-specific long intergenic noncoding RNA that is highly induced in lipopolysaccharide/IFNγ-stimulated macrophages. SIMALR knockdown in lipopolysaccharide/IFNγ stimulated THP1 human macrophages induced apoptosis of inflammatory macrophages, as shown by increased protein expression of cleaved PARP (poly[ADP-ribose] polymerase), caspase 9, caspase 3, and Annexin V+. RNA-sequencing of control versus SIMALR knockdown in lipopolysaccharide/IFNγ-stimulated macrophages showed Netrin-1 ( NTN1 ) to be significantly decreased upon SIMALR knockdown. We confirmed that NTN1 knockdown in lipopolysaccharide/IFNγ-stimulated macrophages induced apoptosis. The SIMALR knockdown-induced apoptotic phenotype was rescued by adding recombinant NTN1. NTN1 promoter-luciferase reporter activity was increased in HEK293T (human embryonic kidney 293) cells treated with lentiviral overexpression of SIMALR . NTN1 promoter activity is known to require HIF1α (hypoxia-inducible factor 1 subunit alpha), and our studies suggest that SIMALR may interact with HIF1α to regulate NTN1 transcription, thereby regulating macrophages apoptosis. SIMALR was found to be expressed in macrophages in human carotid atherosclerotic plaques of symptomatic patients., Conclusions: SIMALR is a nonconserved, human macrophage lncRNA expressed in atherosclerosis that suppresses macrophage apoptosis. SIMALR partners with HIF1α (hypoxia-inducible factor 1 subunit alpha) to regulate NTN1, which is a known macrophage survival factor. This work illustrates the importance of interrogating the functions of human lncRNAs and exploring their translational and therapeutic potential in human atherosclerosis.

Cardiovascular diseases represent a major cause of morbidity and mortality, necessitating research to improve diagnostics, and to discover and test novel preventive and curative therapies, all of which warrant experimental models that recapitulate human disease. The translation of basic science results to clinical practice is a challenging task, in particular for complex conditions such as cardiovascular diseases, which often result from multiple risk factors and comorbidities. This difficulty might lead some individuals to question the value of animal research, citing the translational 'valley of death', which largely reflects the fact that studies in rodents are difficult to translate to humans. This is also influenced by the fact that new, human-derived in vitro models can recapitulate aspects of disease processes. However, it would be a mistake to think that animal models do not represent a vital step in the translational pathway as they do provide important pathophysiological insights into disease mechanisms particularly on an organ and systemic level. While stem cell-derived human models have the potential to become key in testing toxicity and effectiveness of new drugs, we need to be realistic, and carefully validate all new human-like disease models. In this position paper, we highlight recent advances in trying to reduce the number of animals for cardiovascular research ranging from stem cell-derived models to in situ modelling of heart properties, bioinformatic models based on large datasets, and state-of-the-art animal models, which show clinically relevant characteristics observed in patients with a cardiovascular disease. We aim to provide a guide to help researchers in their experimental design to translate bench findings to clinical routine taking the replacement, reduction, and refinement (3R) as a guiding concept., Competing Interests: Conflict of interest: L.B. is supported by unrestricted grants form Astra Zeneca. A.D. is co-founder of InVitroSys GmbH, a start-up developing equipment for biomimetic tissue culture. T.E. is co-founder of EHT Technologies GmbH, a university spin-off providing equipment for the generation of EHT. P.F. is the founder and CEO of Pharmahungary Group, a group of R&D companies. L.F. has received institutional research grants and non-financial support from European Union, British Heart Foundation, Medical Research Council (UK), DFG and several biomedical companies. L.F. is listed as inventor of two patents held by University of Birmingham (Atrial Fibrillation Therapy WO 2015140571 and Markers for Atrial Fibrillation WO 2016012783). L.F. has served on the Roche Advisory Board on the topic New Biomarkers in Atrial Fibrillation. S.H. is independent consultant or receives research grant from to Astra Zeneca, Bayer, Merck, and Pfizer. The APHP, which employs J.-S.H., has received research grants from Bioserenity, Sanofi, Servier and Novo Nordisk. J.-S.H. has received speaker, advisory board or consultancy fees from Amgen, Astra Zeneca, Bayer, Bristol-Myers Squibb, Novartis, Novo Nordisk, and WeHealth. The UMCU, which employs L.W.v.L. has received speaker, advisory board or consultancy fees and/or research grants from Abbott, Vifor, Novartis, Medtronic, Roche, and Sopachem. J.L. has received research grants from Medtronic. P.v.d.M. received consultancy and/or research grants from Vifor Pharma, AstraZeneca, Servier, Novartis, Pfizer, Ionis. R.P. is co-founder of Pluriomics (Ncardia) and River BioMedics. R.S. received speaker fees from Amgen, Recordati and Sanofi and research grants from Sanofi. T.T. filed and licensed patents in the filled of non-coding RNAs. T.T. is founder and shareholder of Cardior Pharmaceuticals, a clinical-stage biotech company. T.T. received support and/or holds advisory seats at Boehringer Ingelheim, Novo Nordisk, Sanofi-Genzyme, Takeda, Amicus Therapeutics. All other authors have declared no conflict of interest., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2022. For permissions, please email: journals.permissions@oup.com.)

Abdominal aortic aneurysms (AAA) is a multifactorial complex disease with life-threatening consequences. While Genome-wide association studies (GWAS) have revealed several single nucleotide polymorphisms (SNPs) located in the genome of individuals with AAA, the link between SNPs with the associated pathological signals, the influence of risk factors on their distribution and their combined analysis is not fully understood. We integrated 86 AAA SNPs from GWAS and clinical cohorts from the literature to determine their phenotypical vulnerabilities and association with AAA risk factors. The SNPs were annotated using snpXplorer AnnotateMe tool to identify their chromosomal position, minor allele frequency, CADD (Combined Annotation Dependent Depletion), annotation-based pathogenicity score, variant consequence, and their associated gene. Gene enrichment analysis was performed using Gene Ontology and clustered using REVIGO. The plug-in GeneMANIA in Cytoscape was applied to identify network integration with associated genes and functions. 15 SNPs affecting 20 genes with a CADD score above ten were identified. AAA SNPs were predominantly located on chromosome 3 and 9. Stop-gained rs5516 SNP obtained high frequency in AAA and associated with proinflammatory and vascular remodeling phenotypes. SNPs presence positively correlated with hypertension, dyslipidemia and smoking history. GO showed that AAA SNPs and their associated genes could regulate lipid metabolism, extracellular matrix organization, smooth muscle cell proliferation, and oxidative stress, suggesting that part of these AAA traits could stem from genetic abnormalities. We show a library of inborn SNPs and associated genes that manifest in AAA. We uncover their pathological signaling trajectories that likely fuel AAA development., (© 2022. The Author(s).)

Objective: The indication for abdominal aortic aneurysm (AAA) repair is based on a diameter threshold. However, mechanical properties, such as peak wall stress (PWS) and peak wall rupture index (PWRI), influence the individual rupture risk. This study aims to correlate biomechanical and geometrical AAA characteristics during aneurysm growth applying a new linear transformation-based comparison of sequential imaging., Methods: Patients with AAA with two sequential computed tomography angiographies (CTA) were identified from a single-center aortic database. Patient characteristics included age, gender, and comorbidities. Semiautomated segmentation of CTAs was performed using Endosize (Therenva) for geometric variables (diameter, neck configuration, α/β angle, and vessel tortuosity) and for finite element method A4 Clinics Research Edition (Vascops) for additional variables (intraluminal thrombus [ILT]), vessel volume, PWS, PWRI). Maximum point coordinates from at least one CTA 6 to 24 months before their final were predicted for the final preoperative CTA using linear transformation along fix and validation points to estimate spatial motion. Pearson's correlation and the t test were used for comparison., Results: Thirty-two eligible patients (median age, 70 years) were included. The annual AAA growth rate was 3.7 mm (interquartile range [IQR], 2.25-5.44; P  < .001) between CTs. AAA (+17%; P  < .001) and ILT (+43%; P  < .001) volume, maximum ILT thickness (+35%; P  < .001), β angle (+1.96°; P  = .017) and iliac tortuosity (+0.009; P  = .012) increased significantly. PWS (+12%; P  = .0029) and PWRI (+16%; P  < .001) differed significantly between both CTAs. Both mechanical parameters correlated most significantly with the AAA volume increase (r = 0.68 [ P  < .001] and r = 0.6 [ P  < .001]). Changes in PWS correlated best with the aneurysm neck configuration. The spatial motion of maximum ILT thickness was 14.4 mm (IQR, 7.3-37.2), for PWS 8.4 mm (IQR, 3.8-17.3), and 11.5 mm (IQR, 5.9-31.9) for PWRI. Here, no significant correlation with any of the aforementioned parameters, patient age, or time interval between CTs were observed., Conclusions: PWS correlates highly significant with vessel volume and aneurysm neck configuration. Spatial motion of maximum ILT thickness, PWS, and PWRI is detectable and predictable and might expose different aneurysm wall segments to maximum stress throughout aneurysm growth. Linear transformation could thus add to patient-specific rupture risk analysis., Clinical Relevance: Abdominal aortic aneurysm rupture risk assessment is a key feature in future individualized therapy approaches for patients, since more and more data are obtained concluding a heterogeneous disease entity that might not be addressed ideally looking only at diameter enlargement. The approach presented in this pilot study demonstrates the feasibility and importance of measuring peak wall stress and rupture risk indices based on predicted and actual position of maximum stress points including intraluminal thrombus., (Copyright © 2022 by the Society for Vascular Surgery. Published by Elsevier Inc.)

In healthy vessels, endothelial cells maintain a stable, differentiated, and growth-arrested phenotype for years. Upon injury, a rapid phenotypic switch facilitates proliferation to restore tissue perfusion. Here we report the identification of the endothelial cell-enriched long non-coding RNA (lncRNA) PCAT19, which contributes to the proliferative switch and acts as a safeguard for the endothelial genome. PCAT19 is enriched in confluent, quiescent endothelial cells and binds to the full replication protein A (RPA) complex in a DNA damage- and cell-cycle-related manner. Our results suggest that PCAT19 limits the phosphorylation of RPA2, primarily on the serine 33 (S33) residue, and thereby facilitates an appropriate DNA damage response while slowing cell cycle progression. Reduction in PCAT19 levels in response to either loss of cell contacts or knockdown promotes endothelial proliferation and angiogenesis. Collectively, PCAT19 acts as a dynamic guardian of the endothelial genome and facilitates rapid switching from quiescence to proliferation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Dissecting the pathways regulating the adaptive immune response in atherosclerosis is of particular therapeutic interest. Here we report that the lipid G-protein coupled receptor GPR55 is highly expressed by splenic plasma cells (PC), upregulated in mouse spleens during atherogenesis and human unstable or ruptured compared to stable plaques. Gpr55 -deficient mice developed larger atherosclerotic plaques with increased necrotic core size compared to their corresponding controls. Lack of GPR55 hyperactivated B cells, disturbed PC maturation and resulted in immunoglobulin (Ig)G overproduction. B cell-specific Gpr55 depletion or adoptive transfer of Gpr55 -deficient B cells was sufficient to promote plaque development and elevated IgG titers. In vitro , the endogenous GPR55 ligand lysophsophatidylinositol (LPI) enhanced PC proliferation, whereas GPR55 antagonism blocked PC maturation and increased their mitochondrial content. Collectively, these discoveries provide previously undefined evidence for GPR55 in B cells as a key modulator of the adaptive immune response in atherosclerosis., Competing Interests: Competing interests The authors declare no conflicts of interests.

Aims: De-differentiation and activation of pro-inflammatory pathways are key transitions vascular smooth muscle cells (SMCs) make during atherogenesis. Here, we explored the upstream regulators of this 'atherogenic transition'., Methods and Results: Genome-wide sequencing studies, including Assay for Transposase-Accessible Chromatin using sequencing and RNA-seq, were performed on cells isolated from both murine SMC-lineage-tracing models of atherosclerosis and human atherosclerotic lesions. At the bulk level, alterations in chromatin accessibility were associated with the atherogenic transitioning of lesional SMCs, especially in relation to genes that govern differentiation status and complement-dependent inflammation. Using computational biology, we observed that a transcription factor previously related to coronary artery disease, Activating transcription factor 3 (ATF3), was predicted to be an upstream regulator of genes altered during the transition. At the single-cell level, our results indicated that ATF3 is a key repressor of SMC transitioning towards the subset of cells that promote vascular inflammation by activating the complement cascade. The expression of ATF3 and complement component C3 was negatively correlated in SMCs from human atherosclerotic lesions, suggesting translational relevance. Phenome-wide association studies indicated that genetic variation that results in reduced expression of ATF3 is correlated with an increased risk for atherosclerosis, and the expression of ATF3 was significantly down-regulated in humans with advanced vascular disease., Conclusion: Our study indicates that the plasticity of atherosclerotic SMCs may in part be explained by dynamic changes in their chromatin architecture, which in turn may contribute to their maladaptive response to inflammation-induced stress., Competing Interests: Conflict of interest: The authors declare that there is no conflict of interest., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

The evolutionary conserved Taurine Upregulated Gene 1 (TUG1) is a ubiquitously expressed gene that is one of the highest expressed genes in human and rodent endothelial cells (ECs). We here show that TUG1 expression decreases significantly in aging mouse carotid artery ECs and human ECs in vitro, indicating a potential role in the aging endothelial vasculature system. We therefore investigated if, and how, TUG1 might function in aging ECs, but despite extensive phenotyping found no alterations in basal EC proliferation, apoptosis, barrier function, migration, mitochondrial function, or monocyte adhesion upon TUG1 silencing in vitro. TUG1 knockdown did slightly and significantly decrease cumulative sprout length upon vascular endothelial growth factor A stimulation in human umbilical vein endothelial cells (HUVECs), though TUG1-silenced HUVECs displayed no transcriptome-wide mRNA expression changes explaining this effect. Further, ectopic expression of the highly conserved and recently discovered 153 amino acid protein translated from certain TUG1 transcript isoforms did not alter angiogenic sprouting in vitro. Our data show that, despite a high expression and strong evolutionary conservation of both the TUG1 locus and the protein sequence it encodes, TUG1 does not seem to play a major role in basic endothelial cell function., Competing Interests: The authors have declared that no competing interest exist

To fulfil its orchestration of immune cell trafficking, a network of chemokines and receptors developed that capitalizes on specificity, redundancy, and functional selectivity. The discovery of heteromeric interactions in the chemokine interactome has expanded the complexity within this network. Moreover, some inflammatory mediators, not structurally linked to classical chemokines, bind to chemokine receptors and behave as atypical chemokines (ACKs). We identified macrophage migration inhibitory factor (MIF) as an ACK that binds to chemokine receptors CXCR2 and CXCR4 to promote atherogenic leukocyte recruitment. Here, we hypothesized that chemokine-chemokine interactions extend to ACKs and that MIF forms heterocomplexes with classical chemokines. We tested this hypothesis by using an unbiased chemokine protein array. Platelet chemokine CXCL4L1 (but not its variant CXCL4 or the CXCR2/CXCR4 ligands CXCL8 or CXCL12) was identified as a candidate interactor. MIF/CXCL4L1 complexation was verified by co-immunoprecipitation, surface plasmon-resonance analysis, and microscale thermophoresis, also establishing high-affinity binding. We next determined whether heterocomplex formation modulates inflammatory/atherogenic activities of MIF. Complex formation was observed to inhibit MIF-elicited T-cell chemotaxis as assessed by transwell migration assay and in a 3D-matrix-based live cell-imaging set-up. Heterocomplexation also blocked MIF-triggered migration of microglia in cortical cultures in situ, as well as MIF-mediated monocyte adhesion on aortic endothelial cell monolayers under flow stress conditions. Of note, CXCL4L1 blocked binding of Alexa-MIF to a soluble surrogate of CXCR4 and co-incubation with CXCL4L1 attenuated MIF responses in HEK293-CXCR4 transfectants, indicating that complex formation interferes with MIF/CXCR4 pathways. Because MIF and CXCL4L1 are platelet-derived products, we finally tested their role in platelet activation. Multi-photon microscopy, FLIM-FRET, and proximity-ligation assay visualized heterocomplexes in platelet aggregates and in clinical human thrombus sections obtained from peripheral artery disease (PAD) in patients undergoing thrombectomy. Moreover, heterocomplexes inhibited MIF-stimulated thrombus formation under flow and skewed the lamellipodia phenotype of adhering platelets. Our study establishes a novel molecular interaction that adds to the complexity of the chemokine interactome and chemokine/receptor-network. MIF/CXCL4L1, or more generally, ACK/CXC-motif chemokine heterocomplexes may be target structures that can be exploited to modulate inflammation and thrombosis., (© 2022. The Author(s).)

Coronary artery disease (CAD) is a complex, multifactorial disease caused, in particular, by inflammation and cholesterol metabolism. At the molecular level, the role of tissue-specific signaling pathways leading to CAD is still largely unexplored. This study relied on two main resources: (1) genes with impact on atherosclerosis/CAD, and (2) liver-specific transcriptome analyses from human and mouse studies. The transcription factor activating transcription factor 3 (ATF3) was identified as a key regulator of a liver network relevant to atherosclerosis and linked to inflammation and cholesterol metabolism. ATF3 was predicted to be a direct and indirect (via MAF BZIP Transcription Factor F (MAFF)) regulator of low-density lipoprotein receptor (LDLR). Chromatin immunoprecipitation DNA sequencing (ChIP-seq) data from human liver cells revealed an ATF3 binding motif in the promoter regions of MAFF and LDLR. siRNA knockdown of ATF3 in human Hep3B liver cells significantly upregulated LDLR expression (p < 0.01). Inflammation induced by lipopolysaccharide (LPS) stimulation resulted in significant upregulation of ATF3 (p < 0.01) and subsequent downregulation of LDLR (p < 0.001). Liver-specific expression data from human CAD patients undergoing coronary artery bypass grafting (CABG) surgery (STARNET) and mouse models (HMDP) confirmed the regulatory role of ATF3 in the homeostasis of cholesterol metabolism. This study suggests that ATF3 might be a promising treatment candidate for lowering LDL cholesterol and reducing cardiovascular risk.

Background and Aims:  The study aimed to assess the potential of proteoglycans (PGs) and collagens as serological biomarkers in the abdominal aortic aneurysm (AAA). Furthermore, we investigated the underlying mechano-biological interactions and signaling pathways., Methods:  Tissue and serum samples from patients with ruptured AAA (rAAA; n  = 29), elective AAA (eAAA; n  = 78), and healthy individuals ( n  = 8) were evaluated by histology, immunohistochemistry, and enzyme-linked immunosorbent assay, and mechanical properties were assessed by tensile tests. Regulatory pathways were determined by membrane-based sandwich immunoassay., Results:  In AAA samples, collagen type I and III (Col1 and Col3), chondroitin sulfate, and dermatan sulfate (DS) were significantly increased compared with controls (3.0-, 3.2-, 1.3-, and 53-fold; p  < 0.01). Col1 and endocan were also elevated in the serum of AAA patients (3.6- and 6.0-fold; p  < 0.01), while DS was significantly decreased (2.5-fold; p  < 0.01). Histological scoring showed increased total PGs and focal accumulation in rAAA compared with eAAA. Tissue β-stiffness was higher in rAAA compared with eAAA (2.0-fold, p  = 0.02). Serum Col1 correlated with maximum tensile force and failure tension ( r  = 0.448 and 0.333; p  < 0.01, and r  = 0.02), tissue endocan correlated with α-stiffness ( r  = 0.340; p  < 0.01). Signaling pathways in AAA were associated with extracellular matrix synthesis and vascular smooth muscle cell proliferation. In particular, Src family kinases and platelet-derived growth factor- and epidermal growth factor-related proteins seem to be involved., Conclusion:  Our findings reveal a structural association between collagen and PGs and their response to changes in mechanical loads in AAA. Particularly Col1 and endocan reflect the mechano-biological conditions of the aortic wall also in the patient's serum and might serve for AAA risk stratification., Competing Interests: None declared., (Thieme. All rights reserved.)

Objective: The purpose of this study was to assess the associations between malignancy, therapeutic regimens, and aorto-iliac aneurysm (i.e., abdominal aortic aneurysm [AAA]) growth rates., Methods: A retrospective single centre analysis identified patients with an AAA plus cancer. Patients who had two or more computed tomography angiograms over six months or more and additional malignancy were included. Clinical data and aneurysm diameters were analysed. AAA growth under cancer therapy (chemotherapy or radiation) was compared with a non-cancer AAA control cohort and to meta-analysis data. Statistics included t tests and a linear regression model with correction for initial aortic diameter and type of treatment., Results: From 2003 to 2020, 217 patients (median age 70 years; 92% male) with 246 aneurysms (58.8% AAA) and 238 malignancies were identified. Prostate (26.7%) and lung (15.7%) cancer were most frequently seen. One hundred and fifty-seven patients (72.3%) received chemotherapy, 105 patients (48.4%) radiation, and 79 (36.4%) both. Annual AAA growth (mean ± standard deviation) was not statistically significantly different for cancer and non-cancer patients (2.0 ± 2.3 vs. 2.8 ± 2.1 mm/year; p = .20). However, subgroup analyses revealed that radiation was associated with a statistically significantly reduced mean aneurysm growth rate compared with cancer patients without radiation (1.1 ± 1.3 vs. 1.6 ± 2.1 mm/year; p = .046) and to the non-cancer control cohort (1.7 ± 1.9 vs. 2.8 ± 2.1 mm/year; p = .007). Administration of antimetabolites resulted in statistically significantly increased AAA growth (+ 0.9 mm/year; p = .011), while topoisomerase inhibitors (- 0.8 mm/year; p = .17) and anti-androgens (- 0.5 mm/year; p = .27) showed a possible trend for reduced growth. Similar observations were noted for iliac aneurysms (n = 85). Additionally, the effects persisted for chemotherapy combinations (2.6 ± 1.4 substances/patient)., Conclusion: Patients with cancer and concomitant aortic aneurysms may require intensified monitoring when undergoing specific therapies, such as antimetabolite treatment, as they may experience an increased aneurysm growth rate. Radiation may be associated with reduced aneurysm growth., (Copyright © 2022 European Society for Vascular Surgery. Published by Elsevier B.V. All rights reserved.)

Background: A significant burden of atherosclerotic disease is driven by inflammation. Recently, microRNAs (miRNAs) have emerged as important factors driving and protecting from atherosclerosis. miR-223 regulates cholesterol metabolism and inflammation via targeting both cholesterol biosynthesis pathway and NF k B signaling pathways; however, its role in atherosclerosis has not been investigated. We hypothesize that miR-223 globally regulates core inflammatory pathways in macrophages in response to inflammatory and atherogenic stimuli thus limiting the progression of atherosclerosis., Methods and Results: Loss of miR-223 in macrophages decreases Abca1 gene and protein expression as well as cholesterol efflux to apoA1 (Apolipoprotein A1) and enhances proinflammatory gene expression. In contrast, overexpression of miR-223 promotes the efflux of cholesterol and macrophage polarization toward an anti-inflammatory phenotype. These beneficial effects of miR-223 are dependent on its target gene, the transcription factor Sp3 . Consistent with the antiatherogenic effects of miR-223 in vitro, mice receiving miR223 -/- bone marrow exhibit increased plaque size, lipid content, and circulating inflammatory cytokines (ie, IL-1β). Deficiency of miR-223 in bone marrow-derived cells also results in an increase in circulating pro-atherogenic cells (total monocytes and neutrophils) compared with control mice. Furthermore, the expression of miR-223 target gene ( Sp3 ) and pro-inflammatory marker ( Il-6 ) are enhanced whereas the expression of Abca1 and anti-inflammatory marker ( Retnla ) are reduced in aortic arches from mice lacking miR-223 in bone marrow-derived cells. In mice fed a high-cholesterol diet and in humans with unstable carotid atherosclerosis, the expression of miR-223 is increased. To further understand the molecular mechanisms underlying the effect of miR-223 on atherosclerosis in vivo, we characterized global RNA translation profile of macrophages isolated from mice receiving wild-type or miR223 -/- bone marrow. Using ribosome profiling, we reveal a notable upregulation of inflammatory signaling and lipid metabolism at the translation level but less significant at the transcription level. Analysis of upregulated genes at the translation level reveal an enrichment of miR-223-binding sites, confirming that miR-223 exerts significant changes in target genes in atherogenic macrophages via altering their translation., Conclusions: Our study demonstrates that miR-223 can protect against atherosclerosis by acting as a global regulator of RNA translation of cholesterol efflux and inflammation pathways.

Aims: Inflammation is a key factor in atherosclerosis. The transcription factor interferon regulatory factor-5 (IRF5) drives macrophages towards a pro-inflammatory state. We investigated the role of IRF5 in human atherosclerosis and plaque stability., Methods and Results: Bulk RNA sequencing from the Carotid Plaque Imaging Project biobank were used to mine associations between major macrophage associated genes and transcription factors and human symptomatic carotid disease. Immunohistochemistry, proximity extension assays, and Helios cytometry by time of flight (CyTOF) were used for validation. The effect of IRF5 deficiency on carotid plaque phenotype and rupture in ApoE-/- mice was studied in an inducible model of plaque rupture. Interferon regulatory factor-5 and ITGAX/CD11c were identified as the macrophage associated genes with the strongest associations with symptomatic carotid disease. Expression of IRF5 and ITGAX/CD11c correlated with the vulnerability index, pro-inflammatory plaque cytokine levels, necrotic core area, and with each other. Macrophages were the predominant CD11c-expressing immune cells in the plaque by CyTOF and immunohistochemistry. Interferon regulatory factor-5 immunopositive areas were predominantly found within CD11c+ areas with a predilection for the shoulder region, the area of the human plaque most prone to rupture. Accordingly, an inducible plaque rupture model of ApoE-/-Irf5-/- mice had significantly lower frequencies of carotid plaque ruptures, smaller necrotic cores, and less CD11c+ macrophages than their IRF5-competent counterparts., Conclusion: Using complementary evidence from data from human carotid endarterectomies and a murine model of inducible rupture of carotid artery plaque in IRF5-deficient mice, we demonstrate a mechanistic link between the pro-inflammatory transcription factor IRF5, macrophage phenotype, plaque inflammation, and its vulnerability to rupture., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Cardiology.)

The nitric oxide-cGMP (NO-cGMP) pathway is of outstanding importance for vascular homeostasis and has multiple beneficial effects in vascular disease. Neointimal hyperplasia after vascular injury is caused by increased proliferation and migration of vascular smooth muscle cells (VSMCs). However, the role of NO-cGMP signaling in human VSMCs in this process is still not fully understood. Here, we investigate the interaction between platelet derived growth factor (PDGF)-signaling, one of the major contributors to neointimal hyperplasia, and the cGMP pathway in vascular smooth muscle, focusing on NO-sensitive soluble guanylyl cyclase (sGC). We show that PDGF reduces sGC expression by activating PI3K and Rac1, which in turn alters Notch ligand signaling. These data are corroborated by gene expression analysis in human atheromas, as well as immunohistological analysis of diseased and injured arteries. Collectively, our data identify the crosstalk between PDGF and NO/sGC signaling pathway in human VSMCs as a potential target to tackle neointimal hyperplasia., (© 2022. The Author(s).)

The pleiotropic benefits of statins may result from their impact on vascular inflammation. The molecular process underlying this phenomenon is not fully elucidated. Here, RNA sequencing designed to investigate gene expression patterns following CD47-SIRPα inhibition identifies a link between statins, efferocytosis, and vascular inflammation. In vivo and in vitro studies provide evidence that statins augment programmed cell removal by inhibiting the nuclear translocation of NFκB1 p50 and suppressing the expression of the critical 'don't eat me' molecule, CD47. Statins amplify the phagocytic capacity of macrophages, and thus the anti-atherosclerotic effects of CD47-SIRPα blockade, in an additive manner. Analyses of clinical biobank specimens suggest a similar link between statins and CD47 expression in humans, highlighting the potential translational implications. Taken together, our findings identify efferocytosis and CD47 as pivotal mediators of statin pleiotropy. In turn, statins amplify the anti-atherosclerotic effects of pro-phagocytic therapies independently of any lipid-lowering effect., Competing Interests: Competing Interests I.L.W. and N.J.L. are co-founders and directors of Bitterroot Bio Incorporated, a cardiovascular company studying macrophage checkpoint inhibition. K.-U.J., Y.K., I.L.W., and N.J.L. have filed a provisional patent (U.S. Application Serial No. 63/106,794): ‘CD47 Blockade and Combination Therapies Thereof For Reduction Of Vascular Inflammation’. The remaining authors declare no competing interests.