Eriksenlaugesen1894

Positron emission tomography (PET) is a quantitative imaging technique that uses molecules labeled with positron-emitting radionuclides to visualize and measure biochemical processes in the tissues of living subjects. In recent years, different PET tracers have been evaluated for their ability to characterize the atherosclerotic process in order to study the activity of the disease. Here, we describe detailed PET methods for preclinical studies of atherosclerosis and summarize the key methodological aspects of PET imaging in clinical studies of atherosclerosis.Atheromatous lesions are characterized by intrusion into the vascular lumen, resulting in morphological changes to the blood compartment and into the vessel wall, resulting in characteristic molecular and cellular signatures in the solid tissue of the intima, tunica media, adventitia and surrounding tissue. Nanoprobes can be easily formulated to provide long blood-pool residence and molecular targeting, facilitating the imaging of atheromatous changes. Detection of nanoprobes can be accomplished by a variety of methods. We focus in this chapter on the use of cross-sectional imaging techniques, computed tomography (CT) and magnetic resonance imaging (MRI), that facilitate in vivo, noninvasive imaging of the vascular morphology and molecular/cellular signatures of the atheroma. The methods described are suitable for use in animal models, although versions of the probes are being readied for clinical trials, potentially facilitating clinical use in the future.The management of cardiovascular conditions will likely be improved by noninvasive in vivo molecular imaging technologies that can provide earlier or more accurate diagnosis. These techniques are already having a positive impact in preclinical research by providing insight into disease pathobiology or efficacy of new therapies. Contrast enhanced ultrasound (CEU) molecular imaging is a technique that relies on the ultrasound detection of targeted microbubble contrast agents to examine molecular or cellular events that occur at the blood pool-endothelial interface. For the most part, targeted contrast agents are composed of encapsulated gas microbubbles (MBs) that are 2-4 μm in diameter, or other acoustically active micro- or nanoparticles. These agents bear several tens of thousands of binding molecules per particle. Because nonadhered agent is cleared rapidly, CEU molecular imaging can be performed in a matter of minutes. MBs are detected using contrast-specific techniques that generate and receive nonlinear signals produced by MB cavitation, thereby increasing signal-to-noise ratio. Dedicated kinetic models for molecular imaging have been generated that permit the elimination of signal from nonadherent agent.Atherosclerosis is characterized by the abundant infiltration of immune cells starting at early stages and progressing to late stages of the disease. The study and characterization of immune cells infiltrating and residing in the aorta has being tackled by several methodologies such as flow cytometry and mass cytometry (CyTOF). Flow cytometry has been primarily used to address the aortic leukocyte composition; however, only a limited number of markers can be analyzed simultaneously. CyTOF started to overcome these limitations by employing rare element-tagged antibodies and combines mass spectrometry with the ease and precision of flow cytometry. CyTOF currently allows for the simultaneous measurement of more than 40 cellular parameters at single-cell resolution.In this chapter, we describe the methodology used to isolate single immune cells from mouse aortas, followed by protocols for flow cytometry and CyTOF for aortic immune cell characterization.The transcriptomic information obtained by single cell RNA sequencing (scRNA-seq) can be supplemented by information on the cell surface phenotype by using oligonucleotide-tagged monoclonal antibodies (scAb-Seq). This is of particular importance in immune cells, where the correlation between mRNA and cell surface expression is very weak. scAb-Seq is facilitated by the availability of commercial antibodies and antibody mixes. Now panels of up to 200 antibodies are available for human and mouse cells. Proteins are detected by antibodies conjugated to a tripartite DNA sequence that contains a primer for amplification and sequencing, a unique oligonucleotide that acts as an antibody barcode and a poly(dA) sequence, simultaneously detecting extension of antibody-specific DNA sequences and cDNAs in the same poly(dT)-primed reaction. For each cell, surface protein expression is captured and sequenced along with the cell's transcriptome. Here, we list the steps needed to produce antibody sequencing data from tissue or blood cells.Recent advances in cardiovascular research have led to a more comprehensive understanding of molecular mechanisms of atherosclerosis. It has become apparent that the disease involves three layers of the arterial wall the intima, the media, and a connective tissue coat termed the adventitia. It is also now appreciated that arteries are surrounded by adipose and neuronal tissues. In addition, adjacent to and within the adventitia, arteries are embedded in a loose connective tissue containing blood vessels (vasa vasora) and lymph vessels, artery-draining lymph nodes and components of the peripheral nervous system, including periarterial nerves and ganglia. During atherogenesis, each of these tissues undergoes marked structural and cellular alterations. We propose that a better understanding of these cell-cell and cell-tissue interactions may considerably advance our understanding of cardiovascular disease pathogenesis. Methods to acquire subcellular optical access to the intact tissues surrounding healthy and diseased arteries are urgently needed to achieve these aims. Tissue clearing is a landmark next-generation, three-dimensional (3D) microscopy technique that allows to image large-scale hitherto inaccessible intact deep tissue compartments. It allows for detailed reconstructions of arteries by a combination of labelling, clearing, advanced microscopies and other imaging and data-analysis tools. Here, we describe two distinct tissue clearing protocols; solvent-based modified three-dimensional imaging of solvent-cleared organs (3DISCO) clearing and another using aqueous-based 2,2'-thiodiethanol (TDE) clearing, both of which complement each other.Although various pro- and anti-inflammatory T cell subsets have been observed in murine and human atherosclerosis, principal issues of T cell immunity remain unanswered Is atherosclerosis progression critically affected by aberrant T cell responses? Are tolerance checkpoints compromised during atherosclerosis progression? Answers to these questions will determine if we are at the cusp of developing T cell-dependent therapeutic strategies. Rapid advances in single cell RNA sequencing (scRNA-seq) and single cell α/β T cell receptor (TCR) (scTCR) sequencing allows to address these issues in unprecedented ways. The majority of T cells recognize peptide antigen-MHC complexes presented by antigen-presenting cells which, in turn, trigger activation and proliferation (clonal expansion) of cognate TCR-carrying T cells. Thus, clonal expansion and their corresponding transcriptome are two similarly important sides of T cell immunity and both will-as hypothesized-affect the outcome of atherosclerosis. Here, we combined scRNA-seq and scTCR-seq in single cells. Moreover, we provide single T cell transcriptomes and TCR maps of three important tissues involved in atherosclerosis This approach is anticipated to address principal questions concerning atherosclerosis autoimmunity that are likely to pave the long sought way to T cell-dependent therapeutic approaches.A major goal of methodologies related to large scale gene expression analyses is to initiate comprehensive information on transcript signatures in single cells within the tissue's anatomy. Until now, this could be achieved in a stepwise experimental approach (1) identify the majority of transcripts in a single cell (single cell transcriptome); (2) provide information on transcripts on multiple cell subtypes in a complex sample (cell heterogeneity); and (3) give information on each cell's spatial location within the tissue (zonation transcriptomics). Such genetic information will allow construction of functionally relevant gene expression maps of single cells of a given anatomically defined tissue compartment and thus pave the way for subsequent analyses, including their epigenetic modifications. Until today these aims have not been achieved in the area of cardiovascular disease research though steps toward these goals become apparent laser capture microdissection (LCM)-based mRNA expression microarrays of atherosclerotic plaques were applied to gain information on local gene expression changes during disease progression, providing limited spatial resolution. Moreover, while LCM-derived tissue RNA extracts have been shown to be highly sensitive and covers a range of 10-16,000 genes per array/small amount of RNA, its original promise to isolate single cells from a tissue section turned out not to be practicable because of the inherent contamination of the cell's RNA of interest with RNA from neighboring cells. Many shortcomings of LCM-based analyses have been overcome using single-cell RNA sequencing (scRNA-seq) technologies though scRNA-seq also has several limitations including low numbers of transcripts/cell and the complete loss of spatial information. Here, we describe a protocol toward combining advantages of both techniques while avoiding their flaws.The low-density lipoprotein receptor (Ldlr) and apolipoprotein E (Apoe) germline knockout (KO) models have provided fundamental insights in lipid and atherosclerosis research for decades. However, testing new candidate genes in these models requires extensive breeding, which is highly time and resource consuming. In this chapter, we provide methods for rapidly modeling hypercholesterolemia and atherosclerosis as well as testing new genes in adult mice through somatic gene editing. Adeno-associated viral (AAV) vectors are exploited to deliver the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing system (AAV-CRISPR) to the liver. This tool enables rapid and efficient editing of lipid- and atherosclerosis-related genes in the liver.In situ hybridization (ISH) is a technique for the detection of the location of RNA within a tissue of interest. This process uses oligonucleotides with complementary sequences to bind to the target RNA, and colorimetric detection to allow for the visualization of this binding. The process of ISH means that the specific location of the RNA in question can be detected, including in which cell types it is present, and the intracellular location. In the case of long noncoding RNA (lncRNA), which do not lead to the production of proteins, ISH is essential for tissue localization. Moreover, RNA abundance is often lower than for protein-coding genes, thus necessitating enhanced detection through double-digoxigenin (DIG) labeling of the probes. Here, we describe the theory and practicalities of performing ISH for lncRNA, with particular reference to vascular tissues.