The initiation of atherosclerosis results from complex interactions of circulating factors and various cell types in the vessel wall, including endothelial cells, lymphocytes, monocytes, and smooth muscle cells (SMCs). Recent reviews highlight the role of activated endothelium and inflammatory cell recruitment in the initiation of and progression of early atherosclerosis. Yet, human autopsy studies, in vitro mechanistic studies, and in vivo correlative data suggest an important role for SMCs in the initiation of atherosclerosis. SMCs are the major producers of extracellular matrix within the vessel wall and in response to atherogenic stimuli can modify the type of matrix proteins produced. In turn, the type of matrix present can affect the lipid content of the developing plaque and the proliferative index of the cells that are adherent to it. SMCs are also capable of functions typically attributed to other cell types. Like macrophages, SMCs can express a variety of receptors for lipid uptake and can form foam-like cells, thereby participating in the early accumulation of plaque lipid. Like endothelial cells, SMCs can also express a variety of adhesion molecules such as vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 to which monocytes and lymphocytes can adhere and migrate into the vessel wall. In addition, through these adhesion molecules, SMCs can also stabilize these cells against apoptosis, thus contributing to the early cellularity of the lesion. Like many cells within the developing plaque, SMCs also produce many cytokines such as PDGF, transforming growth factor-β, IFNγ, and MCP-1, all of which contribute to the initiation and propagation of the inflammatory response to lipid. Recent advances in SMC-specific gene modulation have enhanced our ability to determine the role of SMCs in early atherogenesis.

Background: Rupture and erosion of advanced atherosclerotic lesions with a resultant myocardial infarction or stroke are the leading worldwide cause of death. However, we have a limited understanding of the identity, origin, and function of many cells that make up late-stage atherosclerotic lesions, as well as the mechanisms by which they control plaque stability. Methods: We conducted a comprehensive single-cell RNA sequencing of advanced human carotid endarterectomy samples and compared these with single-cell RNA sequencing from murine microdissected advanced atherosclerotic lesions with smooth muscle cell (SMC) and endothelial lineage tracing to survey all plaque cell types and rigorously determine their origin. We further used chromatin immunoprecipitation sequencing (ChIP-seq), bulk RNA sequencing, and an innovative dual lineage tracing mouse to understand the mechanism by which SMC phenotypic transitions affect lesion pathogenesis. Results: We provide evidence that SMC-specific Klf4- versus Oct4-knockout showed virtually opposite genomic signatures, and their putative target genes play an important role regulating SMC phenotypic changes. Single-cell RNA sequencing revealed remarkable similarity of transcriptomic clusters between mouse and human lesions and extensive plasticity of SMC- and endothelial cell-derived cells including 7 distinct clusters, most negative for traditional markers. In particular, SMC contributed to a Myh11 ^- , Lgals3 ⁺ population with a chondrocyte-like gene signature that was markedly reduced with SMC- Klf4 knockout. We observed that SMCs that activate Lgals3 compose up to two thirds of all SMC in lesions. However, initial activation of Lgals3 in these cells does not represent conversion to a terminally differentiated state, but rather represents transition of these cells to a unique stem cell marker gene–positive, extracellular matrix-remodeling, “pioneer” cell phenotype that is the first to invest within lesions and subsequently gives rise to at least 3 other SMC phenotypes within advanced lesions, including Klf4-dependent osteogenic phenotypes likely to contribute to plaque calcification and plaque destabilization. Conclusions: Taken together, these results provide evidence that SMC-derived cells within advanced mouse and human atherosclerotic lesions exhibit far greater phenotypic plasticity than generally believed, with Klf4 regulating transition to multiple phenotypes including Lgals3 ⁺ osteogenic cells likely to be detrimental for late-stage atherosclerosis plaque pathogenesis.

Rationale:: B cells contribute to atherosclerosis through subset-specific mechanisms. Whereas some controversy exists about the role of B-2 cells, B-1a cells are atheroprotective because of secretion of atheroprotective IgM antibodies independent of antigen. B-1b cells, a unique subset of B-1 cells that respond specifically to T-cell–independent antigens, have not been studied within the context of atherosclerosis. Objective:: To determine whether B-1b cells produce atheroprotective IgM antibodies and function to protect against diet-induced atherosclerosis. Methods and Results:: We demonstrate that B-1b cells are sufficient to produce IgM antibodies against oxidation-specific epitopes on low-density lipoprotein both in vitro and in vivo. In addition, we demonstrate that B-1b cells provide atheroprotection after adoptive transfer into B- and T-cell deficient ( Rag1 ^−/− Apoe ^−/− ) hosts. We implicate inhibitor of differentiation 3 (Id3) in the regulation of B-1b cells as B-cell–specific Id3 knockout mice (Id3 ^BKO Apoe ^−/− ) have increased numbers of B-1b cells systemically, increased titers of oxidation-specific epitope–reactive IgM antibodies, and significantly reduced diet-induced atherosclerosis when compared with Id3 ^WT Apoe ^−/− controls. Finally, we report that the presence of a homozygous single nucleotide polymorphism in ID3 in humans that attenuates Id3 function is associated with an increased percentage of circulating B-1 cells and anti–malondialdehyde-low-density lipoprotein IgM suggesting clinical relevance. Conclusions:: These results provide novel evidence that B-1b cells produce atheroprotective oxidation-specific epitope–reactive IgM antibodies and protect against atherosclerosis in mice and suggest that similar mechanisms may occur in humans.

• B cells regulate atherosclerotic plaque formation through production of antibodies and cytokines, and effects are subset specific (B1 and B2). • Putative human atheroprotective B1 cells function similarly to murine B1 in their spontaneous IgM antibody production. However, marker strategies in identifying human and murine B1 are different. • IgM antibody to oxidation specific epitopes produced by B1 cells associate with human coronary artery disease. • Neoantigen immunization may be a promising strategy for atherosclerosis vaccine development, but further study to determine relevant antigens still need to be done. • B-cell–targeted therapies, used in treating autoimmune diseases as well as lymphoid cancers, might have potential applications in treating cardiovascular diseases. Short- and long-term cardiovascular effects of these agents need to be assessed.

Objective—: Little is known about the role(s) B cells play in obesity-induced metabolic dysfunction. This study used a mouse with B-cell–specific deletion of Id3 (Id3 ^{Bcell KO} ) to identify B-cell functions involved in the metabolic consequences of obesity. Approach and Results—: Diet-induced obese Id3 ^{Bcell KO} mice demonstrated attenuated inflammation and insulin resistance in visceral adipose tissue (VAT), and improved systemic glucose tolerance. VAT in Id3 ^{Bcell KO} mice had increased B-1b B cells and elevated IgM natural antibodies to oxidation-specific epitopes. B-1b B cells reduced cytokine production in VAT M1 macrophages, and adoptively transferred B-1b B cells trafficked to VAT and produced natural antibodies for the duration of 13-week studies. B-1b B cells null for Id3 demonstrated increased proliferation, established larger populations in Rag1 ^−/− VAT, and attenuated diet-induced glucose intolerance and VAT insulin resistance in Rag1 ^−/− hosts. However, transfer of B-1b B cells unable to secrete IgM had no effect on glucose tolerance. In an obese human population, results provided the first evidence that B-1 cells are enriched in human VAT and IgM antibodies to oxidation-specific epitopes inversely correlated with inflammation and insulin resistance. Conclusions—: NAb-producing B-1b B cells are increased in Id3 ^{Bcell KO} mice and attenuate adipose tissue inflammation and glucose intolerance in diet-induced obese mice. Additional findings are the first to identify VAT as a reservoir for human B-1 cells and to link anti-inflammatory IgM antibodies with reduced inflammation and improved metabolic phenotype in obese humans.

Although much of the research on atherosclerosis has focused on the intimal accumulation of lipids and inflammatory cells, there is an increasing amount of interest in the role of the adventitia in coordinating the immune response in atherosclerosis. In this review of the contributions of the adventitia and adventitial lymphocytes to the development of atherosclerosis, we discuss recent research on the formation and structural nature of adventitial immune aggregates, potential mechanisms of crosstalk between the intima, media, and adventitia, specific contributions of B lymphocytes and T lymphocytes, and the role of the vasa vasorum and surrounding perivascular adipose tissue. Furthermore, we highlight techniques for the imaging of lymphocytes in the vasculature.