Next-Generation and Single-Cell Sequencing Approaches to Study Atherosclerosis and Vascular Inflammation Pathophysiology: A Systematic Review

Abstract

Background and AimsAtherosclerosis is a chronic inflammatory disease that remains the leading cause of morbidity and mortality worldwide. Despite decades of research into the development and progression of this disease, current management and treatment approaches remain unsatisfactory and further studies are required to understand the exact pathophysiology. This review aims to provide a comprehensive assessment of currently published data utilizing single-cell and next-generation sequencing techniques to identify key cellular and molecular contributions to atherosclerosis and vascular inflammation.MethodsElectronic searches of Cochrane Central Register of Controlled Trials, MEDLINE, and EMBASE databases were undertaken from inception until February 2022. A narrative synthesis of all included studies was performed for all included studies. Quality assessment and risk of bias analysis was evaluated using the ARRIVE and SYRCLE checklist tools.ResultsThirty-four studies were eligible for narrative synthesis, with 16 articles utilizing single-cell exclusively, 10 utilizing next-generation sequencing and 8 using a combination of these approaches. Studies investigated numerous targets, ranging from exploratory tissue and plaque analysis, cell phenotype investigation and physiological/hemodynamic contributions to disease progression at both the single-cell and whole genome level. A significant area of focus was placed on smooth muscle cell, macrophage, and stem/progenitor contributions to disease, with little focus placed on contributions of other cell types including lymphocytes and endothelial cells. A significant level of heterogeneity exists in the outcomes from single-cell sequencing of similar samples, leading to inter-sample and inter-study variation.ConclusionsSingle-cell and next-generation sequencing methodologies offer novel means of elucidating atherosclerosis with significantly higher resolution than previous methodologies. These approaches also show significant potential for translatability into other vascular disease states, by facilitating cell-specific gene expression profiles between disease states. Implementation of these technologies may offer novel approaches to understanding the disease pathophysiology and improving disease prevention, management, and treatment.Systematic Review Registration:https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021229960, identifier: CRD42021229960.

Single-cell transcriptomics reveals subtype-specific molecular profiles in Nrf2-deficient macrophages from murine atherosclerotic aortas

“…The expression of Cxcl1, Nlrp3, Sdc4, Cxcl16 , and Ccl3 genes characterized both proinflammatory and AIR macrophages (8), but the presence of typical AIR macrophage markers Cd72, Ccrl2, Ctss, Acp5, Mpeg1 ( 8 ) distinguished this population from the proinflammatory subset ( Figure 3E ). Monocyte cluster (8) showed high expression of Plac8, Chil3, Msrb1, Clec4e, and Slpi ( Figure 3E ); HPC/fibrocytes ( 38 ) were enriched in Dcn, Sparc, Gsn, Col3a1, Mgp ( Figure 3E ), while SEM cluster ( 39 ) highly expressed Lum, Runx1, Apoe, Acta2, Ctsb ( Figure 3E ). For cDC1 (8) and cDC2 (8) we found overlapping expression of classical marker genes with higher expression of Naaa, Cst3, Ifi205, Cd24a, H2afz for cDC1 ( Figure 3E ) and Napsa, H2-ab1, H2-eb1, Traf1, Wnt11 for cDC2 ( Figure 3E ).…”

Heterogeneity of macrophages in atherosclerosis revealed by single‐cell RNA sequencing

“…32 However, in recent years, single-cell RNA sequencing (scRNA-seq) has revealed the diversity of macrophage populations within atherosclerotic plaques. 33,34 Using scRNA-seq, atherosclerotic subpopulations and macrophage heterogeneity can be identified, providing much more information about the transcriptomes in individual cells. [35][36][37][38][39] Gene expression and function varies among these subsets, which are involved in atherosclerosis development, stabilization, regression, and rupture of plaque lesions.…”

“…However, in recent years, single‐cell RNA sequencing (scRNA‐seq) has revealed the diversity of macrophage populations within atherosclerotic plaques 33,34 . Using scRNA‐seq, atherosclerotic subpopulations and macrophage heterogeneity can be identified, providing much more information about the transcriptomes in individual cells 35–39 .…”

Identification of Macrophage-related Gene Signature Associated with Unstable Atherosclerotic Plaques through Integrative Multi- omics Analysis

“…Macrophages possess remarkable plasticity and a multitude of biological functions, which has prompted scientists to study their relationship with atherosclerosis for over 50 years, and signi cant discoveries are still being made today [5] . Recent advances in single-cell RNA sequencing technology have allowed scientists to uncover the remarkable diversity of macrophage populations within atherosclerotic plaques [27][28][29][30] . These subpopulations exhibit distinct gene expression and functional characteristics, and they play critical roles in the initiation, progression, stabilization, regression, and rupture of atherosclerotic plaques [31,32] .…”