Human Atherosclerosis Research Articles

GM-CSF-activated STAT5A regulates macrophage functions and inflammation in atherosclerosis.

Inhibition of STAT5 was recently reported to reduce murine atherosclerosis. However, the role of STAT5 isoforms, and more in particular STAT5A in macrophages in the context of human atherosclerosis remains unknown. Here, we demonstrate reciprocal expression regulation of STAT5A and STAT5B in human atherosclerotic lesions. The former was highly upregulated in ruptured over stable plaque and correlated with macrophage presence, a finding that was corroborated by the high chromosomal accessibility of STAT5A but not B gene in plaque macrophages. Phosphorylated STAT5 correlated with macrophages confirming its activation status. As macrophage STAT5 is activated by GM-CSF, we studied the effects of its silencing in GM-CSF differentiated human macrophages. STAT5A knockdown blunted the immune response, phagocytosis, cholesterol metabolism, and augmented apoptosis terms on transcriptional levels. These changes could partially be confirmed at functional level, with significant increases in apoptosis and decreases in lipid uptake and IL-6, IL-8, and TNFa cytokine secretion after STAT5A knockdown. Finally, inhibition of general and isoform A specific STAT5 significantly reduced the secretion of TNFa, IL-8 and IL-10 in ex vivo tissue slices of advanced human atherosclerotic plaques. In summary, we identify STAT5A as an important determinant of macrophage functions and inflammation in the context of atherosclerosis and show its promise as therapeutic target in human atherosclerotic plaque inflammation.

Immune landscape and regulatory mechanisms in human atherosclerotic coronary plaques: Evidence from single-cell and bulk transcriptomics

Atherosclerosis is a chronic immuno-inflammatory disease, however, the immune landscape and regulatory mechanisms have not been clear. We detected seven principal immune cell clusters with distinct phenotypic and spatial characteristics using single-cell RNA-sequencing of aortic immune cells from patients with acute coronary syndrome and stable angina pectoris. Then we acquired 265 differentially expressed immune-related genes and the high scores were mainly found in T cells and monocytes, which were differentially regulated in atherosclerotic coronary plaques. The CCL signaling pathway was the most relevant pattern in the T cells and CCL5-CCR1 and CCL5-CCR5 ligand-receptor pairs played a vital role in the CCL signaling pathway. Further comparative analysis indicated MCH-I signaling was the most relevant pattern in the T cells and HLA ligand-related ligand-receptor pairs played a vital role. Functional analysis of the single-cell and bulk transcriptomics pointed to multiple pathways, such as antigen presentation and immune response. Nineteen common differentially expressed immune-related genes were found in both immune cells and the human peripheral blood mononuclear cells. Nine common differentially expressed transcription factors were differentially expressed in both T cell and monocyte clusters from the coronary plaques and human peripheral blood mononuclear cells and the network demonstrated that CEBPB might play an essential role in the transcriptional regulation of atherosclerosis as a hub transcription factor. The definition of immune cell diversity and heterogeneity by single-cell level analysis of aortic immune cell subsets not only unveils cell-type-specific pathways and new immune mechanisms but also discovers the functional correlation of immune cells in human atherosclerosis. Our findings provide great promise for the discovery of novel molecular mechanisms and precise therapeutic targets for atherosclerosis.

Role of miR-15a-5p and miR-199a-3p in the inflammatory pathway regulated by NF-κB in experimental and human atherosclerosis.

Cardiovascular diseases (CVDs) prevalence has significantly increased in the last decade and atherosclerosis development is the main trigger. MicroRNAs (miRNAs) are non-coding RNAs that negatively regulate gene expression of their target and their levels are frequently altered in CVDs. By RT-qPCR, we analysed miR-9-5p, miR-15a-5p, miR-16-5p and miR-199a-3p levels in aorta from apolipoprotein knockout (ApoE-/- ) mice, an experimental model of hyperlipidemia-induced atherosclerosis, and in human aortic and carotid atherosclerotic samples. By in silico studies, Western blot analysis and immunofluorescence studies, we detected the targets of the altered miRNAs. Our results show that miR-15a-5p and miR-199a-3p are significantly decreased in carotid and aortic samples from patients and mice with atherosclerosis. In addition, we found an increased expression in targets of both miRNAs that participate in the inflammatory pathway of nuclear factor kappa B (NF-κB), such as IKKα, IKKβ and p65. In human vein endothelial cells (HUVECs) and vascular smooth muscle cells (VSMCs), the overexpression of miR-15a-5p or miR-199a-3p decreased IKKα, IKKβ and p65 protein levels as well as NF-κB activation. On the other hand, miR-15a-5p and miR-199a-3p overexpression reduced ox-LDL uptake and the inflammation regulated by NF-κB in VSMCs. Moreover, although miR-15a-5p and miR-199a-3p were significantly increased in exosomes from patients with advanced carotid atherosclerosis, only in the ROC analyses for miR-15a-5p, the area under the curve was 0.8951 with a p value of .0028. Our results suggest that the decrease of miR-199a-3p and miR-15a-5p in vascular samples from human and experimental atherosclerosis could be involved in the NF-κB activation pathway, as well as in ox-LDL uptake by VSMCs, contributing to inflammation and progression atherosclerosis. Finally, miR-15a-5p could be used as a novel diagnostic biomarker for advanced atherosclerosis.

Identification of a PRDM1-regulated T-cell network which controls T-cell driven plaque inflammation in human and mouse atherosclerosis

Identification of a PRDM1-regulated T-cell network which controls T-cell driven plaque inflammation in human and mouse atherosclerosis

Immunopeptidomic analysis of human atherosclerosis identifies novel ApoB100-derived antigenic drivers of atherosclerosis

Immunopeptidomic analysis of human atherosclerosis identifies novel ApoB100-derived antigenic drivers of atherosclerosis

The interleukin-1 receptor type-1 in disturbed flow-induced endothelial mesenchymal activation.

Atherosclerosis is a progressive disease that develops in areas of disturbed flow (d-flow). Progressive atherosclerosis is characterized by bulky plaques rich in mesenchymal cells and high-grade inflammation that can rupture leading to sudden cardiac death or acute myocardial infarction. In response to d-flow, endothelial cells acquire a mesenchymal phenotype through endothelial-to-mesenchymal transition (EndMT). However, the signaling intermediaries that link d-flow to EndMT are incompletely understood. In this study we found that in human atherosclerosis, cells expressing SNAI1 (Snail 1, EndMT transcription factor) were highly expressed within the endothelial cell (EC) layer and in the pre-necrotic areas in unstable lesions, whereas stable lesions did not show any SNAI1 positive cells, suggesting a role for EndMT in lesion instability. The interleukin-1 (IL-1), which signals through the type-I IL-1 receptor (IL-1R1), has been implicated in plaque instability and linked to EndMT formation in vitro. Interestingly, we observed an association between SNAI1 and IL-1R1 within ECs in the unstable lesions. To establish the causal relationship between EndMT and IL-1R1 expression, we next examined IL-1R1 levels in our Cre-lox endothelial-specific lineage tracing mice. IL-1R1 and Snail1 were highly expressed in ECs under atheroprone compared to athero-protective areas, and oscillatory shear stress (OSS) increased IL-1R1 protein and mRNA levels in vitro. Exposure of ECs to OSS resulted in loss of their EC markers and higher induction of EndMT markers. By contrast, genetic silencing of IL-1R1 significantly reduced the expression of EndMT markers and Snail1 nuclear translocation, suggesting a direct role for IL-1R1 in d-flow-induced EndMT. In vivo, re-analysis of scRNA-seq datasets in carotid artery exposed to d-flow confirmed the IL-1R1 upregulation among EndMT population, and in our partial carotid ligation model of d-flow, endothelial cell specific IL-1R1 KO significantly reduced SNAI1 expression. Global inhibition of IL-1 signaling in atherosclerosis as a therapeutic target has recently been tested in the completed CANTOS trial, with promising results. However, the data on IL-1R1 signaling in different vascular cell-types are inconsistent. Herein, we show endothelial IL-1R1 as a novel mechanosensitive receptor that couples d-flow to IL-1 signaling in EndMT.

Seeing beyond apoptosis: ultrastructural aspects of necrosis in human atherosclerosis

In recent years, there has been an explosive growth of research to decipher the pathobiologic relevance of cell death in the development and progression of various cardiovascular disorders such as arterial remodeling and atherosclerosis. High rates of cell death have been reported in animal models, particularly following balloon catheter injury. Also, in humans there is considerable evidence indicating a close connection between cell death and atherosclerosis. In this regard, diverse biochemical and molecular analysis have suggested that intraplaque cells preferentially die by apoptosis, a mode of cell death considered to be active, highly regulated and programmed. In contrast to apoptosis, necrosis has been classically defined as an uncontrolled form of cell death that can occur in response to chemical or physical insults such as trauma, infection, toxins, or lack of blood supply. Necrosis has long been known to be present within atherosclerotic plaques but to date it is still less well understood and characterized than apoptosis. In addition, although electron microscopy (EM) remains essential in cell death research, only a very small proportion of studies deal with the ultrastructural aspects of cell death and/or include EM images to support their findings. As a consequence, many features of cell death modes in human atherosclerosis have not yet been thoroughly investigated and defined. The present study was undertaken to provide an ultrastructural description of the route/s by which intraplaque cells can die also suggesting novel insights for future research.

High-field magnetic resonance microscopy of aortic plaques in a mouse model of atherosclerosis

ObjectivesPre-clinical models of human atherosclerosis are extensively used; however, traditional histological methods do not allow for a holistic view of vascular lesions. We describe an ex-vivo, high-resolution MRI method that allows the 3 dimensional imaging of the vessel for aortic plaque visualization and quantification.Materials and methodsAortas from apolipoprotein-E-deficient (apoE−/−) mice fed an atherogenic diet (group 1) or a control diet (group 2) were subjected to 14 T MR imaging using a 3D gradient echo sequence. The obtained data sets were reconstructed (Matlab), segmented, and analyzed (Avizo). The aortas were further sectioned and subjected to traditional histological analysis (Oil-Red O and hematoxylin staining) for comparison.ResultsA resolution up to 15 × 10x10 μm3 revealed that plaque burden (mm3) was significantly (p < 0.05) higher in group 1 (0.41 ± 0.25, n = 4) than in group 2 (0.01 ± 0.01, n = 3). The achieved resolution provided similar detail on the plaque and the vessel wall morphology compared with histology. Digital image segmentation of the aorta's lumen, plaque, and wall offered three-dimensional visualizations of the entire, intact aortas.Discussion14 T MR microscopy provided histology-like details of pathologically relevant vascular lesions. This work may provide the path research needs to take to enable plaque characterization in clinical applications.

Spatially Resolved Metabolites in Stable and Unstable Human Atherosclerotic Plaques Identified by Mass Spectrometry Imaging.

Impairments in carbohydrate, lipid, and amino acid metabolism drive features of plaque instability. However, where these impairments occur within the atheroma remains largely unknown. Therefore, we sought to characterize the spatial distribution of metabolites within stable and unstable atherosclerosis in both the fibrous cap and necrotic core. Atherosclerotic tissue specimens from 9 unmatched individuals were scored based on the Stary classification scale and subdivided into stable and unstable atheromas. After performing mass spectrometry imaging on these samples, we identified over 850 metabolite-related peaks. Using MetaboScape, METASPACE, and Human Metabolome Database, we confidently annotated 170 of these metabolites and found over 60 of these were different between stable and unstable atheromas. We then integrated these results with an RNA-sequencing data set comparing stable and unstable human atherosclerosis. Upon integrating our mass spectrometry imaging results with the RNA-sequencing data set, we discovered that pathways related to lipid metabolism and long-chain fatty acids were enriched in stable plaques, whereas reactive oxygen species, aromatic amino acid, and tryptophan metabolism were increased in unstable plaques. In addition, acylcarnitines and acylglycines were increased in stable plaques whereas tryptophan metabolites were enriched in unstable plaques. Evaluating spatial differences in stable plaques revealed lactic acid in the necrotic core, whereas pyruvic acid was elevated in the fibrous cap. In unstable plaques, 5-hydroxyindoleacetic acid was enriched in the fibrous cap. Our work here represents the first step to defining an atlas of metabolic pathways involved in plaque destabilization in human atherosclerosis. We anticipate this will be a valuable resource and open new avenues of research in cardiovascular disease.

Lipid-associated macrophages transition to an inflammatory state in human atherosclerosis, increasing the risk of cerebrovascular complications

The immune system is integral to cardiovascular health and disease. Targeting inflammation ameliorates adverse cardiovascular outcomes. Atherosclerosis, a major underlying cause of cardiovascular disease, is conceptualized as lipid-driven inflammation in which macrophages play a nonredundant role. However, evidence emerging so far from single-cell atlases suggests a dichotomy between lipid-associated and inflammatory macrophage states. Here, we present an inclusive reference atlas of human intraplaque immune cell communities. Combining single-cell RNA sequencing (scRNA-seq) of human surgical carotid endarterectomies in a discovery cohort with bulk RNA-seq and immunohistochemistry in a validation cohort (the Carotid Plaque Imaging Project), we reveal the existence of PLIN2hi/TREM1hi macrophages as a Toll-like receptor (TLR)-dependent inflammatory lipid-associated macrophage state linked to cerebrovascular events. Our study shifts the current paradigm of lipid-driven inflammation by providing biological evidence for a pathogenic macrophage transition to an inflammatory lipid-associated phenotype and for its targeting as a new treatment strategy for cardiovascular disease.

#3330 INFLAMMATION SECONDARY TO CHRONIC KIDNEY DISEASE AND PERITONEAL DIALYSIS ACCELERATES ATHEROSCLEROSIS IN A NEW MOUSE MODEL

Abstract Background and Aims Inflammation is an established contributor to accelerated atherosclerosis, which is highly prevalent in people with chronic kidney disease (CKD) and even more so in those treated with peritoneal dialysis (PD). We therefore hypothesised that PD-induced systemic inflammation is essential in the development of atherosclerosis. To study this we developed a novel mouse model of CKD and PD accelerated atherosclerosis. Method We performed a 5/6ths nephrectomy in ApoE deficient “black 6” mice, fed these mice a high-cholesterol diet (HCD), and intraperitoneally administered 3.86% Physioneal daily for 67 days. There were three groups of mice: control (HCD-only), CKD+HCD, and CKD+HCD+PD, with relevant shams. Tissues were harvested twelve weeks after the nephrectomy. Assessments of residual kidney function, atherosclerotic plaque pathology and immunology, and systemic immune responses were performed. Results Atherosclerotic plaque size in the aortic arch was larger in the PD group compared to the control, increasing from 83,779 μm2 ±18,430 to 109,973 μm2 ±25,602 (p = 0.0349), whilst plaque size was unchanged in the CKD group. In contrast, both the CKD and PD groups displayed altered plaque stability. There was less collagen in the plaque vs control, decreasing from 43.54% ±10.65 to 29.71% ±9.43 and 29.73% ±10.18 respectively. Additionally, fibrous cap thickness decreased in both groups vs control. The plaques from only the PD group showed infiltration by T-cells compared to control, with no changes in macrophage content in either the CKD or PD group. In the spleen in the CKD and PD groups, naïve CD4 cells increased and effector CD4 cells decreased compared to control, with the latter change also occurring in the circulation. Central memory CD4 cells, terminally differentiated Th1 cells, Th17 cells, and ‘vascular homing’ cells all increased in the PD group compared to control mice. These homing cells also increased in circulation. The PD group showed decreases in the spleen of regulatory T-cells, effector Th1 cells, and effector CD8 cells. Notably, all of these changes were absent from the mesenteric lymph node. There was a large drop in monocytes in the spleen and a loss of immature and mature dendritic cells in the PD group. Conclusion Combining CKD, a high-cholesterol diet, and exposure to PD fluids in ApoE−/-mice successfully generated a new model for cardiovascular disease in human PD patients. This new model is in a widespread and well-understood mouse line representative of human atherosclerosis. Here we demonstrate the pronounced effects of CKD and PD on the innate and adaptive immune response which increases atherosclerotic burden and leads to larger more vulnerable plaques

Spatial Transcriptional Mapping Reveals Site-Specific Pathways Underlying Human Atherosclerotic Plaque Rupture

BackgroundAtherosclerotic plaque ruptures, triggered by blood flow–associated biomechanical forces, cause most myocardial infarctions and strokes. ObjectivesThis study aims to investigate the exact location and underlying mechanisms of atherosclerotic plaque ruptures, identifying therapeutic targets against cardiovascular events. MethodsHistology, electron microscopy, bulk and spatial RNA sequencing on human carotid plaques were studied in proximal, most stenotic, and distal regions along the longitudinal blood flow direction. Genome-wide association studies were used to examine heritability enrichment and causal relationships of atherosclerosis and stroke. Associations between top differentially expressed genes (DEGs) and preoperative and postoperative cardiovascular events were examined in a validation cohort. ResultsIn human carotid atherosclerotic plaques, ruptures predominantly occurred in the proximal and most stenotic regions but not in the distal region. Histologic and electron microscopic examination showed that proximal and most stenotic regions exhibited features of plaque vulnerability and thrombosis. RNA sequencing identified DEGs distinguishing the proximal and most stenotic regions from the distal region which were deemed as most relevant to atherosclerosis-associated diseases as shown by heritability enrichment analyses. The identified pathways associated with the proximal rupture-prone regions were validated by spatial transcriptomics, firstly in human atherosclerosis. Of the 3 top DEGs, matrix metallopeptidase 9 emerged particularly because Mendelian randomization suggested that its high circulating levels were causally associated with atherosclerosis risk. ConclusionsOur findings show plaque site–specific transcriptional signatures associated with proximal rupture-prone regions of carotid atherosclerotic plaques. This led to the geographical mapping of novel therapeutic targets, such as matrix metallopeptidase 9, against plaque rupture.

HDL apoprotein immunization induces T cell-mediated venulitis and inflammation in aorta

The hypothesis that atherosclerotic processes are mostly caused by immune (autoimmune) mechanisms has recently been gaining attraction. At the same time, the autoimmune hypothesis of atherogenesis has not become generally accepted and requires additional evidence. Previously, we were able to induce changes in the aortic wall similar to those observed in the early stages of human atherosclerosis, and also to produce visceral obesity in normocholesterolaemic Wistar rats by a single immunization with human native high- or low-density lipoproteins. We also found that the immune response to native human HDL causes atherosclerosis-like lesions in the rabbit aorta, such as adipocyte and chondrocyte metaplasia, proteoglycan deposits, and leukocyte infiltration. Atherosclerosis-like lesions developed in the aorta of hnHDL-immunized rabbits against a background of normal blood LDL-cholesterol level. Thus, an immune response against HDL or LDL may be an independent cause of atherogenesis. The aim of this study was to test whether immunization with human HDL apoproteins (apoA1 and apoE proteins) would induce atherosclerosis-like lesions in the aorta of normocholesterolemic Wistar rats. HDL apoproteins were isolated from human or rat plasma. Wistar rats (n = 5) aged 2 months were used for immunization with human HDL apoproteins. HDL apoproteins were administered as a single intradermal injection of 100 mg per rat in incomplete Freund’s adjuvant. Control rats were injected subcutaneously with incomplete Freund’s adjuvant (n = 5). Rats were dissected 25 weeks after immunization. Rat aorta sections were stained with hematoxylin and eosin for light microscopy. T lymphocytes infiltration was determined by immunohistochemical staining with FITC-labeled antibodies specific to rat CD3. CD3 + T lymphocytes were detected using an Olympus BX53 fluorescent microscope. The level of antibodies to human and rat HDL apoproteins was determined by indirect enzyme-linked immunosorbent assay. Immunization with HDL apoproteins induced a T cell mediated immune response without production of autoantibodies to HDL apoproteins. The aortic intima and adventitia were infiltrated with T lymphocytes in rats immunized with HDL apoproteins. Pronounced T lymphocytic infiltration was found in all layers of the vein wall in rats immunized with human HDL apoproteins. Thus, immunization with HDL apoproteins causes T cell mediated inflammation of the aorta and venulitis.

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

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. 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. 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. 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.

Abstract 701: Atherosclerosis Is A Smooth Muscle Cell-driven Tumor-like Disease

Background: Atherosclerosis, the leading cause of cardiovascular disease (CVD), is a chronic inflammatory disease involving pathological activation of multiple cell types, such as immunocytes (e.g., macrophage, T cells), smooth muscle cells (SMCs), endothelial cells, etc. Multiple lines of evidence have suggested that SMC “phenotypic switching” plays a central role in atherosclerosis development and complications. Yet, how SMCs and SMC-derived cells (SDCs) modulate atherosclerosis progression and clinical CVD remains poorly understood. Methods: We applied SMC lineage tracing mice and implemented comprehensive molecular and cellular biology, histological, computational, and pharmacological studies to reveal how tumor cell-like activities of SDCs drive the development of atherosclerosis. Results: Oxidative DNA damage occurs during SMC phenotypic switching and increases with the progression of atherosclerosis. Extensive DNA damage-induced genomic instability accumulates in both mouse and human atherosclerosis, especially in SDCs. Indeed, SDCs in atherosclerosis harbor multiple tumor cell-like features, including escape of replicative senescence, clonogenicity, invasive capacity, and ex vivo formation of three-dimensional (3D) spheroids like cancer stem cells. Multiple cancer-associated signaling pathways (e.g., p53, TNFa, NFkB, VEGF) are activated in SDCs. SMC-specific expression of oncogenic Kras G12D accelerates SMC phenotypic switching and exacerbates atherosclerosis. Moreover, we provide proof of concept in mouse models that niraparib, an anti-cancer drug targeting DNA damage repair, attenuates atherosclerosis progression and regresses lesions in advanced disease. Conclusions: Our studies in human and mouse models provide the first systematic evidence that SDCs in atherosclerosis share extensive commonalities with tumor, which deepens our understanding of the disease and opens prospects for novel targeted strategies to prevent and treat atherosclerotic CVD.

Abstract 147: Spatially Resolved Metabolites In Stable And Unstable Human Atherosclerotic Plaques Identified By Mass Spectrometry Imaging

Impairments in carbohydrate, lipid, and amino acid metabolism drive features of plaque instability. However, where these impairments occur within the atheroma remains largely unknown. Therefore, we sought to characterize the spatial distribution of metabolites within stable and unstable atherosclerosis in both the fibrous cap and necrotic core. Atherosclerotic tissue specimens were scored based on the Stary classification scale and subdivided into stable and unstable atheromas. After performing mass spectrometry imaging (MSI) on these samples, we identified over 850 metabolite-related peaks. Using MetaboScape, METASPACE, and HMDB, we confidently annotated 170 of these metabolites and found over 60 of these were different between stable and unstable atheromas. We then integrated these results with an RNA-sequencing dataset comparing stable and unstable human atherosclerosis. Upon integrating our MSI results with the RNA-seq dataset, we discovered that pathways related to lipid metabolism and long-chain fatty acids were enriched in stable plaques, whereas reactive oxygen species, aromatic amino acid, and tryptophan metabolism were increased in unstable plaques. Acylcarnitines and acylglycines were increased in stable plaques whereas tryptophan metabolites were enriched in unstable plaques. Evaluating spatial differences in stable plaques revealed lactic acid in the necrotic core, whereas pyruvic acid was elevated in the fibrous cap. In unstable plaques, 5-hydroxyindole-acetic acid was enriched in the fibrous cap. Our work herein represents the first step to defining an atlas of metabolic pathways involved in plaque destabilization in human atherosclerosis. We anticipate this will be a valuable resource and open new avenues of research in cardiovascular disease.

Abstract 465: Integrative Single-cell Meta-analysis Reveals Disease-relevant Vascular Cell States and Markers in Human Atherosclerosis

Introduction: Atherosclerosis is a chronic inflammatory process driven by plaque formation in the major elastic arteries leading to reduced blood flow, coronary artery disease (CAD), myocardial infarction and stroke. Several single-cell RNA-seq (scRNA-seq) studies have highlighted lineage-specific transcriptomic signatures in CAD-relevant tissues; however, there remains variability on the reported cell phenotypes in humans. We evaluated the overall hypothesis that distinct smooth muscle cell (SMC) phenotypes characterized from scRNA-seq data contribute to human atherosclerotic lesion formation. Methods: Here we meta-analyzed scRNA-seq human data from four individual studies to establish a comprehensive map of cell diversity in human atherosclerosis. We applied standardized QC, processing, and integration benchmarking to harmonize datasets and generate an atlas of 118,578 high-quality cells. Results: Using this atlas we characterize vascular and immune cell diversity during atherosclerosis by identifying 100 consensus marker genes across 42 annotated cell types/subtypes. We provide insights into SMC phenotypic modulation through pseudotime, transcription factor activity inference and cell-cell communication analyses. By integrating genome-wide association study (GWAS) data we identify etiologic cell types for subclinical and clinical CAD traits, which uncovered a critical role for modulated SMC phenotypes in CAD and coronary artery calcification. Finally, we identified candidate markers of synthetic and osteochondrogenic human SMCs (e.g., LTBP1 and CRTAC1 ) that may serve as proxies of atherosclerosis progression and provide validation through bulk transcriptomics, proteomics, and immunofluorescence. Conclusions: Together, this represents an important step towards creating a unified cellular map of atherosclerosis to inform cell-specific mechanistic and translational studies of cardiovascular diseases.

Abstract 157: Inkiln is a Novel Long Noncoding RNA Promoting Vascular Smooth Muscle Inflammation Via Scaffolding Mkl1 and Usp10

Activation of vascular smooth muscle cells (VSMCs) inflammation is critical in initiation and progression of vascular disease. However, the role of human-specific long noncoding RNAs (lncRNAs) in VSMC inflammation is largely unknown. We performed Bulk RNA-seq in differentiated human VSMCs and revealed a novel human-specific lncRNA called IN flammatory M K L1 I nteracting L ong N oncoding RNA ( INKILN ). INKILN is barely expressed in contractile VSMCs or healthy vessels and induced in human atherosclerosis and abdominal aortic aneurysm. INKILN is transcriptionally activated through p65/NF-κB pathway. INKILN activates proinflammatory gene expression in cultured human VSMCs and in ex vivo cultured vessels. Mechanistically, INKILN physically binds to and stabilizes MKL1 protein, a key activator of VSMC inflammation through the p65/NF-κB pathway. In the IL1β-activated VSMCs, depletion of INKILN blocks the nuclear localization of both p65 and MKL1, and abolishes the physical interaction between these two proteins, resulting in the reduced transactvity of p65/NF-κB. Further, INKILN knockdown enhances MKL1 ubiquitination, likely through the reduced physical interaction with the deubiquitinating enzyme USP10. The in vivo study of INKILN has utilized a Bacterial Artificial Chromosome (BAC) INKILN -Transgenic (Tg) mouse line, due to the lack of non-conserved INKILN gene in the mouse genome. In the ligation-injured carotid arteries of BAC INKILN -Tg mice, human lncRNA INKILN is induced and has exacerbated neointimal formation. In conclusion, these findings elucidate an important pathway of VSMC inflammation involving an INKILN /MKL1/USP10 regulatory axis. Human BAC Tg mice offer a novel and physiologically relevant approach for investigating human-specific lncRNAs under vascular disease conditions.

Splicing of ultraconserved poison exon elements in RNA binding proteins reprograms the T effector immune response

Abstract Our current understanding of pathways governing the strength and longevity of T cell mediated immune responses primarily focuses on transcriptional and epigenetic changes. However, our prior work demonstrated that optimal T cell effector function is also dependent on RNA binding proteins (RBPs) that carry out post-transcriptional RNA splicing and polyadenylation. Here, we find that a specific family of RBPs, serine- and arginine-rich (SR) RBPs, are significantly upregulated by T cell activation and required for CD8 +T cell mediated responses. SR transcripts contain ultraconserved poison exon (PE) elements that are alternatively spliced and introduce premature stop codons that trigger nonsense-mediated decay. We show that differential splicing of specific RBP PE regions is both induced by TCR stimulation and required for CD8 +T cell activation in both human and mouse. Further, we demonstrate that PEs are cell-state associated by using single-cell RNA sequencing analysis of splicing changes across mouse thymic T cell subsets, in CD8 +T cells over the course of an acute Listeria infection, as well as in human lymphoblastic leukemia, atherosclerosis, and melanoma patient T cells. CRISPR-Cas9 mediated deletion of the PE element of SR-like RBP Tra2β in mouse T effector cells during influenza infection resulted in a significant increase in proliferating and surviving T cells and altered the splicing pattern of critical cell survival, proliferation, and adhesion genes. In total, our work demonstrates a fundamental role for alternatively spliced ultraconserved elements in defining T cell states, controlling T effector-mediated immunity, and altering the T cell response in pathologic conditions. This work was supported in part by NIH grants AI136955 and R21AI139891 awarded to A.T.V, institutional support, and Boehringer Ingelheim endowed Chair in Immunology.

Stabilization of vulnerable plaque in the ACS patient: evidence from HUYGENS studies.

The introduction of PCSK9 inhibitors in addition to statin therapy allowed better control of LDL-C plasma levels with a subsequent reduction of cardiovascular events. Human atherosclerosis has been previously considered an irreversible condition; studies firstly based on angiography imaging and secondly with intra-coronary imaging-mainly IVUS based-have demonstrated that lipid-lowering therapy based on statins can stabilize and even reduce atherosclerotic burden of coronary circulation. While plaque stabilization and/or reduction with PCSK9 inhibitors have already been demonstrated in the GLAGOV study, the HUYGENS study showed a positive effect not only on atherosclerotic burden but also on plaque phenotype, with an increased FCT, decrease in maximum lipid arch, and reduction of macrophages infiltration. Further studies need to assess the clinical impact of the reduction of plaques displaying high-risk features with PCSK9 inhibitors.