Rationale: Atherosclerotic lesions are known for their cellular heterogeneity, yet the molecular complexity within the cells of human plaques has not been fully assessed. Objective: Using single-cell transcriptomics and chromatin accessibility, we gained a better understanding of the pathophysiology underlying human atherosclerosis. Methods and Results: We performed single-cell RNA and single-cell ATAC sequencing on human carotid atherosclerotic plaques to define the cells at play and determine their transcriptomic and epigenomic characteristics. We identified 14 distinct cell populations including endothelial cells, smooth muscle cells, mast cells, B cells, myeloid cells, and T cells and identified multiple cellular activation states and suggested cellular interconversions. Within the endothelial cell population, we defined subsets with angiogenic capacity plus clear signs of endothelial to mesenchymal transition. CD4 ⁺ and CD8 ⁺ T cells showed activation-based subclasses, each with a gradual decline from a cytotoxic to a more quiescent phenotype. Myeloid cells included 2 populations of proinflammatory macrophages showing IL (interleukin) 1B or TNF (tumor necrosis factor) expression as well as a foam cell-like population expressing TREM2 (triggering receptor expressed on myeloid cells 2) and displaying a fibrosis-promoting phenotype. ATACseq data identified specific transcription factors associated with the myeloid subpopulation and T cell cytokine profiles underlying mutual activation between both cell types. Finally, cardiovascular disease susceptibility genes identified using public genome-wide association studies data were particularly enriched in lesional macrophages, endothelial, and smooth muscle cells. Conclusions: This study provides a transcriptome-based cellular landscape of human atherosclerotic plaques and highlights cellular plasticity and intercellular communication at the site of disease. This detailed definition of cell communities at play in atherosclerosis will facilitate cell-based mapping of novel interventional targets with direct functional relevance for the treatment of human disease.

Background— Mast cells are major effector cells in allergy and host defense responses. Their increased number and state of activation in perivascular tissue during atherosclerosis may point to a role in cardiovascular disorders. In the present study, we investigated the contribution of perivascular mast cells to atherogenesis and plaque stability in apolipoprotein E–deficient mice. Methods and Results— We show here that episodes of systemic mast cell activation during plaque progression in mice leads to robust plaque expansion. Targeted activation of perivascular mast cells in advanced plaques sharply increases the incidence of intraplaque hemorrhage, macrophage apoptosis, vascular leakage, and CXCR2/VLA-4–mediated recruitment of leukocytes to the plaque. Importantly, treatment with the mast cell stabilizer cromolyn does prevent all the adverse phenomena elicited by mast cell activation. Conclusions— This is the first study to demonstrate that mast cells play a crucial role in plaque progression and destabilization in vivo. We propose that mast cell stabilization could be a new therapeutic approach to the prevention of acute coronary syndromes.

Myocardial infarction (MI) triggers an intense inflammatory response that is associated with infarct expansion and is detrimental for cardiac function. Interleukin (IL)-1β and IL-18 are key players in this response and are controlled by the NLRP3-inflammasome. In the current study, we therefore hypothesized that selective inhibition of the NLRP3-inflammasome reduces infarct size and preserves cardiac function in a porcine MI model. Thirty female landrace pigs were subjected to 75 min transluminal balloon occlusion and treated with the NLRP3-inflammasome inhibitor MCC950 (6 or 3 mg/kg) or placebo for 7 days in a randomized, blinded fashion. After 7 days, 3D-echocardiography was performed to assess cardiac function and Evans blue/TTC double staining was executed to assess the area at risk (AAR) and infarct size (IS). The IS/AAR was lower in the 6 mg/kg group (64.6 ± 8.8%, P = 0.004) and 3 mg/kg group (69.7 ± 7.2%, P = 0.038) compared with the control group (77.5 ± 6.3%). MCC950 treatment markedly preserved left ventricular ejection fraction in treated animals (6 mg/kg 47 ± 8%, P = 0.001; 3 mg/kg 45 ± 7%, P = 0.031; control 37 ± 6%). Myocardial neutrophil influx was attenuated in treated compared with non-treated animals (6 mg/kg 132 ± 72 neutrophils/mm², P = 0.035; 3 mg/kg 207 ± 210 neutrophils/mm², P = 0.5; control 266 ± 158 neutrophils/mm²). Myocardial IL-1β levels were dose-dependently reduced in treated animals. NLRP3-inflammasome inhibition reduces infarct size and preserves cardiac function in a randomized, blinded translational large animal MI model. Hence, NLRP3-inflammasome inhibition may have therapeutic potential in acute MI patients.

Growth differentiation factor (GDF) 15 is a member of the transforming growth factor β (TGF-β) superfamily, which operates in acute phase responses through a currently unknown receptor. Elevated GDF-15 serum levels were recently identified as a risk factor for acute coronary syndromes. We show that GDF-15 expression is up-regulated as disease progresses in murine atherosclerosis and primarily colocalizes with plaque macrophages. Hematopoietic GDF-15 deficiency in low density lipoprotein receptor^−/− mice led to impaired initial lesion formation and increased collagen in later lesions. Although lesion burden in GDF-15^−/− chimeras was unaltered, plaques had reduced macrophage infiltrates and decreased necrotic core formation, all features of improved plaque stability. In vitro studies pointed to a TGFβRII-dependent regulatory role of GDF-15 in cell death regulation. Importantly, GDF-15^−/− macrophages displayed reduced CCR2 expression, whereas GDF-15 promoted macrophage chemotaxis in a strictly CCR2- and TGFβRII-dependent manner, a phenomenon which was not observed in G protein–coupled receptor kinase 2^+/− macrophages. In conclusion, GDF-15 deletion has a beneficial effect both in early and later atherosclerosis by inhibition of CCR2-mediated chemotaxis and by modulating cell death. Our study is the first to identify GDF-15 as an acute phase modifier of CCR2/TGFβRII-dependent inflammatory responses to vascular injury.