Atherosclerosis Is a Smooth Muscle Cell-Driven Tumor-Like Disease.

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 KrasG12D 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.

Mustn1 is a smooth muscle cell-secreted microprotein that modulates skeletal muscle extracellular matrix composition.

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.

High-Dimensional Single-Cell Multimodal Landscape of Human Carotid Atherosclerosis.

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.

Suppression of IL-1ß promotes beneficial accumulation of fibroblast-like cells in atherosclerotic plaques in clonal hematopoiesis.

Clonal hematopoiesis (CH) is an independent risk factor for atherosclerotic cardiovascular disease. Murine models of CH suggest a central role of inflammasomes and IL-1ß 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-1ß 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 Jak2VF or Tet2 CH treated with IL-1ß 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-1ß antibody resulted in thinner fibrous caps. Conversely, genetic inactivation of Jak2VF 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.

The challenges of research data management in cardiovascular science: a DGK and DZHK position paper-executive summary.

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.

Utilization of an Artery-on-a-Chip to Unravel Novel Regulators and Therapeutic Targets in Vascular Diseases.

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.

ADAMTS-7 Modulates Atherosclerotic Plaque Formation by Degradation of TIMP-1.

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.

The circular RNA Ataxia Telangiectasia Mutated regulates oxidative stress in smooth muscle cells in expanding 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 (cATM) was upregulated in human AAA specimens, in AAA-derived SMCs, and serum samples collected from aneurysm patients. In primary aortic SMCs, cATM increased upon angiotensin II and doxorubicin stimulation, while its silencing triggered apoptosis. Higher cATM levels made AAA-derived SMCs less vulnerable to oxidative stress, compared with control SMCs. These data suggest that cATM contributes to elicit an adaptive oxidative-stress response in SMCs and provides a reliable AAA disease signature.

Leukocytes carrying Clonal Hematopoiesis of Indeterminate Potential (CHIP) Mutations invade Human Atherosclerotic Plaques.

Background: Leukocyte progenitors derived from clonal hematopoiesis of undetermined potential (CHIP) are associated with increased cardiovascular events. However, the prevalence and functional relevance of CHIP in coronary artery disease (CAD) are unclear, and cells affected by CHIP have not been detected in human atherosclerotic plaques. Methods: CHIP mutations in blood and tissues were identified by targeted deep-DNA-sequencing (DNAseq: coverage >3,000) and whole-genome-sequencing (WGS: coverage >35). CHIP-mutated leukocytes were visualized in human atherosclerotic plaques by mutaFISH™. Functional relevance of CHIP mutations was studied by RNAseq. Results: DNAseq of whole blood from 540 deceased CAD patients of the Munich cardIovaScular StudIes biObaNk (MISSION) identified 253 (46.9%) CHIP mutation carriers (mean age 78.3 years). DNAseq on myocardium, atherosclerotic coronary and carotid arteries detected identical CHIP mutations in 18 out of 25 mutation carriers in tissue DNA. MutaFISH™ visualized individual macrophages carrying DNMT3A CHIP mutations in human atherosclerotic plaques. Studying monocyte-derived macrophages from Stockholm-Tartu Atherosclerosis Reverse Networks Engineering Task (STARNET; n=941) by WGS revealed CHIP mutations in 14.2% (mean age 67.1 years). RNAseq of these macrophages revealed that expression patterns in CHIP mutation carriers differed substantially from those of non-carriers. Moreover, patterns were different depending on the underlying mutations, e.g. those carrying TET2 mutations predominantly displayed upregulated inflammatory signaling whereas ASXL1 mutations showed stronger effects on metabolic pathways. Conclusions: Deep-DNA-sequencing reveals a high prevalence of CHIP mutations in whole blood of CAD patients. CHIP-affected leukocytes invade plaques in human coronary arteries. RNAseq data obtained from macrophages of CHIP-affected patients suggest that pro-atherosclerotic signaling differs depending on the underlying mutations. Further studies are necessary to understand whether specific pathways affected by CHIP mutations may be targeted for personalized treatment.

Vascular smooth muscle-specific YAP/TAZ deletion triggers aneurysm development in mouse aorta.

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.

Apolipoprotein E (ApoE) Rescues the Contractile Smooth Muscle Cell Phenotype in Popliteal Artery Aneurysm Disease.

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.

High-Dimensional Single-Cell Multimodal Landscape of Human Carotid Atherosclerosis.

Background: Atherosclerotic plaques are complex tissues composed of a heterogeneous mixture of cells. However, we have limited understanding of the comprehensive transcriptional and phenotypical landscape of the cells within these lesions. Methods: To characterize the landscape of human carotid atherosclerosis in greater detail, we combined cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and single-cell RNA sequencing (scRNA-seq) 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 distinct cell populations each having a unique multi-omic signature, including macrophages, T cells, NK cells, mast cells, B cells, plasma cells, neutrophils, dendritic cells, endothelial cells, fibroblasts, and smooth muscle cells (SMCs). Within the macrophage populations, we identified 2 proinflammatory subsets that were enriched in IL1B or C1Q expression, 2 distinct TREM2 positive foam cell subsets, one of which also expressed inflammatory genes, as well as subpopulations displaying a proliferative gene expression signature and one expressing SMC-specific genes and upregulation of fibrotic pathways. An in-depth characterization uncovered several subsets of SMCs and fibroblasts, including a SMC-derived foam cell. We localized this foamy SMC to the deep intima of coronary atherosclerotic lesions. Using CITE-seq data, we also developed the first flow cytometry panel, using cell surface proteins CD29, CD142, and CD90, to isolate SMC-derived cells from lesions. Last, we found that the proportion of efferocytotic macrophages, classically activated endothelial cells, contractile and modulated SMC-derived cell types were reduced, and inflammatory SMCs were enriched in plaques of clinically symptomatic vs. 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. This facilitates both the mapping of cardiovascular disease susceptibility loci to specific cell types as well as the identification of novel molecular and cellular therapeutic targets for treatment of the disease.

Targeting long non-coding RNA NUDT6 enhances smooth muscle cell survival and limits vascular disease progression.

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.

INKILN is a Novel Long Noncoding RNA Promoting Vascular Smooth Muscle Inflammation via Scaffolding MKL1 and USP10.

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-1ß-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.

Endothelial microRNAs and long noncoding RNAs in cardiovascular ageing.

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.

A comparative gene expression matrix in Apoe-deficient mice identifies unique and atherosclerotic disease stage-specific gene regulation patterns in monocytes and macrophages.

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.

Mast cells participate in smooth muscle cell reprogramming and atherosclerotic plaque calcification.

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.

Rupture risk parameters upon biomechanical analysis independently change from vessel geometry during abdominal aortic aneurysm growth.

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/ß 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), ß 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.