Microanatomy of the Human Atherosclerotic Plaque by Single-Cell Transcriptomics

RATIONALE: Atherosclerotic lesions are known for their cellular heterogeneity, yet the molecular complexity within the cells of human plaques has not been fully assessed. OBJECTIVE: Using single-cell transcriptomics and chromatin accessibility, we gained a better understanding of the pathophysiology underlying human atherosclerosis. METHODS AND RESULTS: We performed single-cell RNA and single-cell ATAC sequencing on human carotid atherosclerotic plaques to define the cells at play and determine their transcriptomic and epigenomic characteristics. We identified 14 distinct cell populations including endothelial cells, smooth muscle cells, mast cells, B cells, myeloid cells, and T cells and identified multiple cellular activation states and suggested cellular interconversions. Within the endothelial cell population, we defined subsets with angiogenic capacity plus clear signs of endothelial to mesenchymal transition. CD4+ and CD8+ T cells showed activation-based subclasses, each with a gradual decline from a cytotoxic to a more quiescent phenotype. Myeloid cells included 2 populations of proinflammatory macrophages showing IL (interleukin) 1B or TNF (tumor necrosis factor) expression as well as a foam cell-like population expressing TREM2 (triggering receptor expressed on myeloid cells 2) and displaying a fibrosis-promoting phenotype. ATACseq data identified specific transcription factors associated with the myeloid subpopulation and T cell cytokine profiles underlying mutual activation between both cell types. Finally, cardiovascular disease susceptibility genes identified using public genome-wide association studies data were particularly enriched in lesional macrophages, endothelial, and smooth muscle cells. CONCLUSIONS: This study provides a transcriptome-based cellular landscape of human atherosclerotic plaques and highlights cellular plasticity and intercellular communication at the site of disease. This detailed definition of cell communities at play in atherosclerosis will facilitate cell-based mapping of novel interventional targets with direct functional relevance for the treatment of human diseas

Intersecting single-cell transcriptomics and genome-wide association studies identifies crucial cell populations and candidate genes for atherosclerosis

Genome-wide association studies (GWASs) have discovered hundreds of common genetic variants for atherosclerotic disease and cardiovascular risk factors. The translation of susceptibility loci into biological mechanisms and targets for drug discovery remains challenging. Intersecting genetic and gene expression data has led to the identification of candidate genes. However, previously studied tissues are often non-diseased and heterogeneous in cell composition, hindering accurate candidate prioritization. Therefore, we analysed single-cell transcriptomics from atherosclerotic plaques for cell-type-specific expression to identify atherosclerosis-associated candidate gene-cell pairs.\nWe applied gene-based analyses using GWAS summary statistics from 46 atherosclerotic and cardiovascular disease, risk factors, and other traits. We then intersected these candidates with single-cell RNA sequencing (scRNA-seq) data to identify genes specific for individual cell (sub)populations in atherosclerotic plaques. The coronary artery disease (CAD) loci demonstrated a prominent signal in plaque smooth muscle cells (SMCs) (SKI, KANK2, and SORT1) P-adj. = 0.0012, and endothelial cells (ECs) (SLC44A1, ATP2B1) P-adj. = 0.0011. Finally, we used liver-derived scRNA-seq data and showed hepatocyte-specific enrichment of genes involved in serum lipid levels.\nWe discovered novel and known gene-cell pairs pointing to new biological mechanisms of atherosclerotic disease. We highlight that loci associated with CAD reveal prominent association levels in mainly plaque SMC and EC populations. We present an intuitive single-cell transcriptomics-driven workflow rooted in human large-scale genetic studies to identify putative candidate genes and affected cells associated with cardiovascular traits. Collectively, our workflow allows for the identification of cell-specific targets relevant for atherosclerosis and can be universally applied to other complex genetic diseases and traits.Biopharmaceutic

Confocal-based fluorescence fluctuation spectroscopy with a SPAD array detector

The combination of confocal laser-scanning microscopy (CLSM) and fluorescence fluctuation spectroscopy (FFS) is a powerful tool in studying fast, sub-resolution biomolecular processes in living cells. A detector array can further enhance CLSM-based FFS techniques, as it allows the simultaneous acquisition of several samples–essentially images—of the CLSM detection volume. However, the detector arrays that have previously been proposed for this purpose require tedious data corrections and preclude the combination of FFS with single-photon techniques, such as fluorescence lifetime imaging. Here, we solve these limitations by integrating a novel single-photon-avalanche-diode (SPAD) array detector in a CLSM system. We validate this new implementation on a series of FFS analyses: spot-variation fluorescence correlation spectroscopy, pair-correlation function analysis, and image-derived mean squared displacement analysis. We predict that the unique combination of spatial and temporal information provided by our detector will make the proposed architecture the method of choice for CLSM-based FFS

The changing landscape of the vulnerable plaque: a call for fine-tuning of preclinical models

For decades, the pathological definition of the vulnerable plaque led to invaluable insights into the mechanisms that underlie myocardial infarction and stroke. Beyond plaque rupture, other mechanisms, such as erosion, may elicit thrombotic events underlining the complexity and diversity of the atherosclerotic disease. Novel insights, based on single-cell transcriptomics and other “omics” methods, provide tremendous opportunities in the ongoing search for cell-specific determinants that will fine-tune the description of the thrombosis prone lesion. It coincides with an increasing awareness that knowledge on lesion characteristics, cell plasticity and clinical presentation of ischemic cardiovascular events have shifted over the past decades. This shift correlates with an observed changes of cell composition towards phenotypical stabilizing of human plaques. These stabilization features and mechanisms are directly mediated by the cells present in plaques and can be mimicked in vitro via primary plaque cells derived from human atherosclerotic tissues. In addition, the rapidly evolving of sequencing technologies identify many candidate genes and molecular mechanisms that may influence the risk of developing an atherosclerotic thrombotic event - which bring the next challenge in sharp focus: how to translate these cell-specific insights into tangible functional and translational discoveries

Early cognitive and emotional outcome after stroke is independent of discharge destination

Background and purpose: Cognitive and emotional problems occur frequently after stroke. Patients with minor stroke are more likely to be discharged home. This paper compares early cognitive and emotional outcomes in patients discharged home after stroke versus patients discharged to inpatient rehabilitation, and examines the effect of cognitive and emotional outcomes on long-term participation. Methods: In this multicenter prospective cohort study, patients with stroke were assessed at two months with the Hospital Anxiety and Depression Scale (HADS), the Checklist for Cognitive and Emotional Consequences following Stroke (CLCE-24) and the Montreal Cognitive Assessment (MoCA). One year post stroke, participation was assessed with the Restriction subscale of the Utrecht Scale for Evaluation of Rehabilitation—Participation (USER-P Restriction). Results: The study included 332 patients. Two months post stroke, anxiety and cognitive problems were equally prevalent among patients discharged home (n = 243; 73%) and patients discharged to inpatient rehabilitation (n = 89; 27%) (HADS-A = 4.8 ± 3.9 versus 4.6 ± 4.0, p = 0.747; MoCA < 26: 66.7% versus 70.8%, p = 0.477; CLCE-cognition = 3.0 ± 2.9 versus 3.3 ± 2.8, p = 0.499). Depressive symptoms were less severe in patients discharged home (HADS-D = 4.3 ± 3.9 versus 5.5 ± 3.8, p = 0.010). In patients discharged home, cognitive complaints were predictive of long-term participation (B = − 2.03; 95% CI − 3.15, − 0.90), while cognitive or emotional outcomes were not predictive in patients discharged to inpatient rehabilitation. Conclusions: Cognitive and emotional problems at two months post stroke were comparable between patients discharged home and those discharged to inpatient rehabilitation. For patients discharged home, cognitive complaints were predictive of long-term participation

Practice variation in the informed consent procedure for thrombolysis in acute ischemic stroke: a survey among neurologists and neurology residents

Background: Obtaining informed consent for intravenous thrombolysis in acute ischemic stroke can be challenging, and little is known about if and how the informed consent procedure is performed by neurologists in clinical practice. This study examines the procedure of informed consent for intravenous thrombolysis in acute ischemic stroke in high-volume stroke centers in the Netherlands. Methods: In four high volume stroke centers, neurology residents and attending neurologists received an online questionnaire concerning informed consent for thrombolysis with tissue-type plasminogen activator (tPA). The respondents were asked to report their usual informed consent practice for tPA treatment and their considerations on whether informed consent should be obtained. Results: From the 203 invited clinicians, 50% (n = 101) completed the questionnaire. One-third of the neurology residents (n = 21) and 21% of the neurologists (n = 8) reported that they always obtain informed consent for tPA treatment. If a patient is not capable of providing informed consent, 30% of the residents (n = 19) reported that they start tPA treatment without informed consent. In these circumstances, 53% of the neurologists (n = 20) reported that the resident under their supervision would start tPA treatment without informed consent. Most neurologists (n = 21; 55%) and neurology residents (n = 45; 72%) obtained informed consent within one minute. None of the respondents used more than five minutes for informed consent. Important themes regarding obtaining informed consent for treatment were patients’ capacity, and medical, ethical and legal considerations. Conclusion: The current practice of informed consent for thrombolysis in acute ischemic stroke varies among neurologists and neurology residents. If informed consent is obtained, most clinicians stated to obtain informed consent within one minute. In the future, a shortened information provision process may be applied, making a shift from informed consent to informed refusal, while still considering the patient’s capacity, stroke severity, and possible treatment delays

Study of Thermophysical Properties of Silver Nanofluids by ISS-HD, Hot Ball and IPPE Techniques

© 2015, Springer Science+Business Media New York. In this work, the impulsive stimulated scattering technique, in a heterodyne diffraction detection configuration (ISS-HD), was used to study the dependence of the speed of sound and the thermal diffusivity on the concentration of silver nanoparticles in water, to which also d-glucose and carboxymethyl cellulose were added, in order to reduce sedimentation. The ISS-HD results, which show a slight increase of thermal diffusivity with increasing concentration, were cross-validated with results obtained by the inverse photopyroelectric method and the hot ball technique.status: publishe

Mapping genes to cardiovascular susceptibility loci at a single-cell resolution

To date, genome-wide association studies (GWAS) have identified hundreds of risk loci for coronary artery disease (CAD), and other cardiometabolic diseases and traits. However, identifying the key genes for atherosclerotic disease in these loci remains challenging. Here, we systematically mapped 14 GWAS and leveraged transcriptomics of advanced atherosclerotic plaques (AP) at a single-cell resolution.We isolated viable, nucleated single-cells from plaques of 3 carotid endarterectomy patients using enzymatic digestion and fluorescence-activated cell sorting. We applied a CEL-seq2/SORT-seq protocol and the Seurat pipeline for single-cell RNA sequencing (scRNAseq) and cell identification, respectively. Next we annotated public GWAS data of cardiovascular diseases, and cardiometabolic traits using FUMA, which is based on LD clumping, physical location, regulatory and transcriptomic data.Using scRNAseq we identified 11 cellular clusters in AP, and integrated these data to map 1,336 loci across 14 cardiometabolic GWAS. For CAD 105 mapped genes in 35 established loci were differentially expressed between cellular clusters. Some of these loci harboured upto 10 differentially expressed genes, highly expressed in endothelial cells, mast cells, and smooth muscle cells. Notably, some CAD genes are almost exclusively expressed in a specific cell: in the NOS3 locus, KCNH2 is highly expressed in mast cells, whereas NOS3 itself, but also AMPD2 (SORT1 locus) are highly expressed in endothelial cells.We systematically mapped and annotated risk loci, and integrated this at a single-cell resolution with transcriptomics from AP. We identified specific genes and cellular clusters relevant to atherosclerotic plaques development and progression, informative for future mechanistic studies.Biopharmaceutic

Microanatomy of advanced human atherosclerotic plaques through single-cell transcriptomics

Atherosclerotic lesions are known for their complex and diverse cellular heterogeneity, yet the exact cellular composition of human plaques remains unclear. Here we aim to provide a comprehensive overview of the cellular content of advanced atherosclerotic lesions using single-cell RNA sequencing (scRNAseq), to increase our understanding of the pathophysiologic processes underlying atherosclerosis.Advanced carotid plaques were obtained from 3 male endarterectomy patients. Plaques were enzymatically digested and live cells were sorted using FACS. Subsequently, we performed scRNAseq on a total of 3456 plaque derived cells.Unsupervised clustering revealed 11 distinct cell populations, including macrophages, CD4+and CD8+T-cells, B-cells, mast cells, endothelial cells, and smooth muscle cells. Interestingly, we were able to distinguish multiple subclusters for the smooth muscle cells, macrophages, and T-cells. We observed a contractile and synthetic smooth muscle cell cluster and three different macrophage clusters, including inflammatory, wound healing and antigen presenting macrophages. Finally, CD8+T-cells could be subdivided into three clusters based on their activation state, with a clear division between cytolytic effectors and quiescent memory CD8+T-cells. We developed a technique to perform scRNAseq on advanced human atherosclerotic lesions to unravel the cellular landscape within the plaques. We now provide, for the first time, an explicit expression profile of the different cells and their subtypes in atherosclerosis.Biopharmaceutic