Translational opportunities of single-cell biology in atherosclerosis

Abstract

The advent of single-cell biology opens a new chapter for understanding human biological processes and for diagnosing, monitoring, and treating disease. This revolution now reaches the field of cardiovascular disease (CVD). New technologies to interrogate CVD samples at single-cell resolution are allowing the identification of novel cell communities that are important in shaping disease development and direct towards new therapeutic strategies. These approaches have begun to revolutionize atherosclerosis pathology and redraw our understanding of disease development. This review discusses the state-of-the-art of single-cell analysis of atherosclerotic plaques, with a particular focus on human lesions, and presents the current resolution of cellular subpopulations and their heterogeneity and plasticity in relation to clinically relevant features. Opportunities and pitfalls of current technologies as well as the clinical impact of single-cell technologies in CVD patient care are highlighted, advocating for multidisciplinary and international collaborative efforts to join the cellular dots of CVD.

Keywords: Atherosclerosis; Endothelial cell; Lymphocyte; Macrophage; Single-cell biology; Smooth muscle cell.

Graphical Abstract

Single-cell biology is bringing new clinical meaning to patient heterogeneity in many disciplines. Atlases of the cellular building blocks of the human atherosclerotic plaque have so far shown that: (i) cellular identity is overall preserved, albeit overlapping transcriptional programmes are activated; (ii) changes in cellular cluster abundance appear between healthy and diseased vascular states; (iii) renewed evidence emerged for a role of T cells in human cardiovascular disease (CVD); and (iv) macrophage heterogeneity supports targeting inflammation and lipids while sparing protective subsets. Vascular single-cell biology has clear translational implications for CVD in terms of identification of the molecular pathways of disease resistance vs. disease propensity and genetic risk, guidance in designing new therapies, vaccines and repurposing drugs for CVD, the study of the therapy-induced adaptation of plaque and circulating cells in clinical trials, improved modelling of human CVD through the availability of metagenomic data sets, and advances in patient selection and stratification. GMZB, Granzyme B; Lef1, lymphoid enhancer binding factor 1; Prf1 Perforin 1; Trem2, triggering receptor expressed on myeloid cells 2.

Figure 1

The basis of cellular heterogeneity in atherosclerosis There are three main determinants of cellular heterogeneity in arteries and atherosclerosis. (i) Cell origin. Arteries have different ontogeny. They originate from the neural crest (carotid and proximal aorta), the proepicardium (coronary arteries) or the mesoderma (rest of the body). Also, macrophages are either resident, or increasingly bone marrow-derived with age. (ii) Cell geography. Cell location imprints cells, either via exposure to different shear stress and other haemodynamic forces, as is the case with ECs, or through cell residence within distinct niches in health and disease. Macrophages have distinct phenotypes in the intima or adventitia or respond to intraplaque events such as haemorrhage or lipid accumulation. (iii) Cell plasticity. EndoMT is characterized by a downregulation of EC-specific gene expression and/or the full disappearance of EC fate marker genes along with the appearance of gene expression programmes associated with other cell types, including fibroblasts, SMCs and macrophages, among others. Often such ‘foreign’ gene expression is associated with activation of EC proliferation and migration and loss of the protective quiescent metabolic state as well as of the ability to exert normal EC function, such as a response to blood flow, regulation of permeability, and antioxidant capacity. These changes in EC gene expression show a remarkable degree of plasticity and can be temporary activated shortly after myocardial infarction, or turn into a permanent EndoMT. A number of pathologic factors including disturbed flow, oxidative stress, hypoxia, and inflammation (e.g. activation of endothelial TGFβ or IL-beta signalling) can initiate EndMT. Pro-atherogenic cues trigger SMC phenotypic modulation and atherosclerotic plaque investment by oligoclonal expansion. Plasticity between different SMC-derived states suggests that SMC phenotypic states might be niche-dependent, transitory, and/or interconvertible. A macrophage-to-mesenchymal transition has also been described.

Figure 2

Cellular populations and plasticity in atherosclerosis. Cellular diversity uncovered by scRNA-seq analysis in human and murine atherosclerosis with focus on consensus populations found in both species. Macrophages are key inflammatory immune cells in atherosclerotic plaques. Four macrophage populations have been identified with different functional features, including: Trem2^hi macrophages, resident-like macrophages, and inflammatory macrophages. Aortic macrophages identified by scRNA-seq can have different origins and derive from infiltrating monocytes, proliferation of embryonically derived macrophages, and lastly from SMCs and fibroblasts obtaining macrophage features. The greatest cellular diversity is found among T cells. Multiple CD4 and CD8 effector and central memory (eff mem) and cytotoxic subsets have been identified that likely contribute to atheroprogression. Regulatory T cells (Tregs) have known anti-inflammatory and atherosclerotic properties. CXCR6⁺ CD4⁺ T cells have a multi-lineage signature and can likely differentiate into other subtypes. Contrasting to T-cell diversity, only one B-cell population has been identified in human atherosclerotic plaques with yet unknown function. EC and SMC undergo various phenotypic transitions likely stage- and micro-environment dependent within the plaque. They can display either atheroprotective or atheropromoting phenotypes. SMCs (top left, adapted from Worssam and Jorgensen) in atherosclerotic lesions undergo progressive cell transitions with loss of the expression profile of contractile cells found in healthy arteries. Instead, SMC-derived cells adopt a range of cell states, displaying some aspects of fibroblasts, mesenchymal cells, and chondrocytes. Evidence for a transient, intermediate cell state with stem-cell, endothelial, and monocyte features (termed SEM)^, and transition of SMC towards a macrophage-like state has been provided by lineage tracing in mouse models. GMZB, Granzyme B; Lef1, Lymphoid Enhancer Binding Factor 1; Prf1, Perforin 1; Trem2, Triggering Receptor Expressed On Myeloid Cells 2.

Figure 3

An iterative and multidisciplinary technical pipeline for scRNA-seq of human atherosclerosis. Samples from patients and healthy individuals can be processed, sequenced, and the resulting raw data filtered and normalized prior to organization into clusters to identify potential cell types involved in the disease. After obtaining raw sequencing data, subsequent bioinformatic steps involve quality controls (e.g. RNA degradation, unmapped reads or mitochondrial genes, or technical errors including cell doublets), normalization of data, removal of poorly sequenced cells, and possibly imputation of empty reads. Additional information, e.g. molecular pathways, differential gene expression, and networks, can be obtained. Novel findings can be transferred to scientific and clinical stakeholders, or used to optimize patient selection, specimen sampling and analysis.

Figure 4

Translational opportunities for single-cell biology. Single-cell data sets allow a multitude of opportunities to better understand and define cardiovascular disease. Single-cell data flows will contribute to the translation from the laboratory to the cardiovascular disease clinics across several avenues. Firstly, they will inform the discovery of novel disease-associated therapeutic targets and drug repositioning. Secondly, they will transform our ability to model human disease in vivo and in vitro, by defining similarities and differences between human and experimental disease irrespective of species and at the single-cell level, and by defining cell phenotyping in situ for in vitro modelling. Finally, vascular single-cell biology will resolve heterogenous atherosclerotic syndromes in endotypes driven by specific cellular processes, identify cellular factors driving patient heterogeneity, and identify residual risk after optimal therapy.