BACKGROUND: Atherosclerotic plaques are complex tissues composed of a heterogeneous mixture of cells. However, our understanding of the comprehensive transcriptional and phenotypic landscape of the cells within these lesions is limited. METHODS: To characterize the landscape of human carotid atherosclerosis in greater detail, we combined cellular indexing of transcriptomes and epitopes by sequencing and single-cell RNA sequencing to classify all cell types within lesions (n=21; 13 symptomatic) to achieve a comprehensive multimodal understanding of the cellular identities of atherosclerosis and their association with clinical pathophysiology. RESULTS: We identified 25 cell populations, each with a unique multiomic signature, including macrophages, T cells, NK (natural killer) cells, mast cells, B cells, plasma cells, neutrophils, dendritic cells, endothelial cells, fibroblasts, and smooth muscle cells (SMCs). Among the macrophages, we identified 2 proinflammatory subsets enriched in IL-1B (interleukin-1B) or C1Q expression, 2 TREM2-positive foam cells (1 expressing inflammatory genes), and subpopulations with a proliferative gene signature and SMC-specific gene signature with fibrotic pathways upregulated. Further characterization revealed various subsets of SMCs and fibroblasts, including SMC-derived foam cells. These foamy SMCs were localized in the deep intima of coronary atherosclerotic lesions. Utilizing cellular indexing of transcriptomes and epitopes by sequencing data, we developed a flow cytometry panel, using cell surface proteins CD29, CD142, and CD90, to isolate SMC-derived cells from lesions. Lastly, we observed reduced proportions of efferocytotic macrophages, classically activated endothelial cells, and contractile and modulated SMC-derived cells, while inflammatory SMCs were enriched in plaques of clinically symptomatic versus asymptomatic patients. CONCLUSIONS: Our multimodal atlas of cell populations within atherosclerosis provides novel insights into the diversity, phenotype, location, isolation, and clinical relevance of the unique cellular composition of human carotid atherosclerosis. These findings facilitate both the mapping of cardiovascular disease susceptibility loci to specific cell types and the identification of novel molecular and cellular therapeutic targets for the treatment of the disease.

Over the past decades, obesity has become a rising health problem as the accessibility to high calorie, low nutritional value food has increased. Research shows that some bioactive components in fruits and vegetables, such as carotenoids, could contribute to the prevention and treatment of obesity. Some of these carotenoids are responsible for vitamin A production, a hormone-like vitamin with pleiotropic effects in mammals. Among these effects, vitamin A is a potent regulator of adipose tissue development, and is therefore important for obesity. This review focuses on the role of the provitamin A carotenoid β-carotene in human health, emphasizing the mechanisms by which this compound and its derivatives regulate adipocyte biology. It also discusses the physiological relevance of carotenoid accumulation, the implication of the carotenoid-cleaving enzymes, and the technical difficulties and considerations researchers must take when working with these bioactive molecules. Thanks to the broad spectrum of functions carotenoids have in modern nutrition and health, it is necessary to understand their benefits regarding to metabolic diseases such as obesity in order to evaluate their applicability to the medical and pharmaceutical fields.

The β-carotene oxygenase 1 (BCO1) is the enzyme responsible for the cleavage of β-carotene to retinal, the first intermediate in vitamin A formation. Preclinical studies suggest that BCO1 expression is required for dietary β-carotene to affect lipid metabolism. The goal of this study was to generate a gene therapy strategy that over-expresses BCO1 in the adipose tissue and utilizes the β-carotene stored in adipocytes to produce vitamin A and reduce obesity. We generated a novel adipose-tissue-specific, adeno-associated vector to over-express BCO1 (AT-AAV-BCO1) in murine adipocytes. We tested this vector using a unique model to achieve β-carotene accumulation in the adipose tissue, in which Bco1^−/− mice were fed β-carotene. An AT-AAV over-expressing green fluorescent protein was utilized as control. We evaluated the adequate delivery route and optimized cellular and organ specificity, dosage, and exposure of our vectors. We also employed morphometric analyses to evaluate the effect of BCO1 expression in adiposity, as well as HPLC and mass spectrometry to quantify β-carotene and retinoids in tissues, including retinoic acid. AT-AAV-BCO1 infusions in the adipose tissue of the mice resulted in the production of retinoic acid, a vitamin A metabolite with strong effects on gene regulation. AT-AAV-BCO1 treatment also reduced adipose tissue size and adipocyte area by 35% and 30%, respectively. These effects were sex-specific, highlighting the complexity of vitamin A metabolism in mammals. The over-expression of BCO1 through delivery of an AT-AAV-BCO1 leads to the conversion of β-carotene to vitamin A in adipocytes, which subsequently results in reduction of adiposity. These studies highlight for the first time the potential of adipose tissue β-carotene as a target for BCO1 over-expression in the reduction of obesity.

BACKGROUND: Vascular smooth muscle cells (VSMCs) play a central role in atherosclerosis by undergoing phenotypic modulation from a quiescent, contractile state to a range of synthetic phenotypes, including fibroblast-like, macrophage-like, and lipid-laden foam cell–like states. However, a comprehensive multimodal characterization and understanding of the transcriptional programs driving these transitions remain incomplete. METHODS: To comprehensively define the phenotypic diversity of VSMCs during atherosclerosis progression, we performed in-depth profiling using cellular indexing of transcriptomes and epitopes by sequencing and bulk RNA sequencing in a VSMC-lineage–tracing atherosclerotic mouse model. Insights from these data sets guided the design of targeted in vitro experiments to investigate candidate regulatory mechanisms. RESULTS: Single-cell multiomics revealed extensive cellular heterogeneity within atherosclerotic plaques, including a rare population of VSMC-derived macrophage-like cells, whose presence was confirmed by histological analysis. These studies also identified a large population of VSMC-derived foam cells that exhibited activation of gene programs associated with lipid metabolism, proliferation, and tumor-like features. The transcription factor BHLHE40 (basic helix-loop-helix family member e40) emerged as a candidate regulator of this phenotypic transition, with elevated expression and activity in VSMC-derived foam cells during disease progression and expression in modulated VSMC in human carotid atherosclerosis. Functional knockdown of Bhlhe40 reprogrammed immune, cell cycle, and lipid homeostasis genes in cultured VSMC and suppressed VSMC phenotypic switching and foam cell characteristics, consistent with a potential regulatory role in VSMC modulation. CONCLUSIONS: These findings advance our understanding of VSMC phenotypic modulation in atherosclerosis and implicate BHLHE40 as a candidate transcriptional regulator of this process. Elucidating mechanisms governing VSMC plasticity may offer new therapeutic opportunities to reduce cardiovascular risk by targeting disease-driving cellular transitions.

BACKGROUND: Atherosclerosis involves complex interactions between lipids, immune cells, vascular smooth muscle cells, and fibroblasts within the arterial wall. While significant advances in single-cell technologies have shed light on the roles of immune cells and vascular smooth muscle cells in plaque development, fibroblasts remain underexplored, leaving critical gaps in understanding their contributions to disease progression and plaque stability. Comprehensive characterization of fibroblast phenotypes in atherosclerosis is essential to unravel their diverse functions and to distinguish between subsets that may play protective versus pathogenic roles in the disease process. METHODS: Here, we utilized cellular indexing of transcriptomes and epitopes by sequencing to comprehensively profile fibroblast diversity in a mouse model of atherosclerosis. Mice were fed an atherogenic diet for 0, 8, 19, and 26 weeks, representing distinct stages of disease progression, enabling a detailed phenotypic characterization of fibroblasts throughout the course of atherosclerosis development. RESULTS: We identified 4 distinct fibroblast subpopulations, including a myofibroblast population closely resembling vascular smooth muscle cell–derived chondromyocytes. The proportions of these fibroblast subsets exhibited a modest decline as atherosclerosis progressed. Through multimodal analysis, we identified CD26 (cluster of differentiation) as a highly expressed and specific marker for one of these fibroblast subpopulations, distinguishing it from other subsets. Using a combination of flow cytometry and immunohistochemistry, we demonstrated that CD26 ⁺ fibroblasts predominantly reside in the adventitia of healthy arteries. During atherosclerosis progression, these cells expand into the intima and primarily localize within the fibrous cap of the lesion. CONCLUSIONS: Our multiomic analysis highlights the phenotypic diversity and dynamic changes of fibroblasts during atherosclerosis progression. Among these, CD26 ⁺ fibroblasts emerge as a distinct subpopulation that expands within atherosclerotic lesions and may play a critical role in promoting plaque stability through their migration into the fibrous cap.

BACKGROUND: Smooth muscle cell (SMC)-derived cells form the bulk of cells in atherosclerotic lesions and modulate lesion stability and cardiovascular disease outcomes. Unfolded protein response (UPR) markers, thin fibrous caps, and inflammation correlate with human lesion instability and rupture. In mice, UPR drives macrophage and endothelial apoptosis and inflammation, but its impact on lesion stability through SMC modulation is debated. The UPR protein PERK (protein kinase RNA-like ER kinase) was recently shown to drive SMC modulation in vivo, suggesting that depletion of SMC Perk may regulate lesion stability. METHODS: SMC Perk was deleted from SMC-lineage-traced adult Ldlr ^−/− hypercholesterolemic mice. Lesions were scored for features of lesion stability and analyzed for differential expression at the single-cell level. Perk knockdown in primary murine SMCs was used to study Perk’s effect on SMC modulation in vitro. Human carotid lesions were assessed for UPR markers through scRNA-seq, bulk RNA-seq, and Xenium spatial transcriptomics. RESULTS: SMC Perk deletion in adult atherogenic mice did not affect weight gain or serum cholesterol levels. Lesions from Perk knockout mice resembled Perk WT counterparts with similar progression, lesion stability features, and cell populations. Scoring of UPR markers and differential expression analysis found little UPR activity in SMC and smooth muscle-derived cell populations. Perk was not required for in vitro SMC modulation in atherogenic conditions. No correlation was found between UPR markers and lesion stability or symptomatic clinical presentation in human carotid lesions, and UPR markers were expressed primarily in infiltrating leukocytes rather than in SMCs and stromal cells. CONCLUSIONS: SMC Perk UPR does not play a significant role in atherosclerotic SMC modulation, disease progression, or features of lesion stability in mice. Similarly, expression of markers of the Perk UPR pathway in humans does not correlate with human carotid lesion stability or clinical presentation.