Project description:Regression of atherosclerosis is an important clinical goal, however the pathways that mediate the resolution of atherosclerotic inflammation and reversal of plaques are poorly understood. Regulatory T cells (Tregs) have been shown to be atheroprotective, however numbers of these immunosuppressive cells decrease with disease progression. Using multiple independent mouse models of atherosclerosis regression, we demonstrate that an increase in plaque Tregs is a common signature of regressing plaques. To test if Tregs are required for the resolution of atherosclerotic inflammation and plaque regression during lipid-lowering therapy, we combined CD25 monoclonal antibody (PC61 mAb)-mediated Treg depletion with single-cell RNA-sequencing of immune cells in the plaque and conventional analyses of atherosclerosis. Single cell RNA-sequencing revealed that Tregs from aortic plaques shared some similarity with splenic Tregs, but were distinct from skin and colon Tregs supporting recent findings of tissue-dependent Treg heterogeneity. Furthermore, Tregs from progressing plaques expressed markers of natural Tregs derived from the thymus, whereas Tregs in regressing plaques lacked Nrp1 and Helios expression, suggesting that they are induced in the periphery during lipid lowering. Treatment of atherosclerotic mice with PC61 mAb effectively depleted Tregs in the blood and peripheral tissues, including plaques, and blocked the regression of atherosclerosis induced by apoB anti-sense oligonucleotides. Morphometric analyses revealed that control antibody-treated mice showed a 40% decrease in plaque burden and macrophage content under regression conditions, whereas PC61 mAb-treated mice showed no change in plaque size or inflammatory cell content compared to baseline. Moreover, Treg depletion enhanced inflammatory signaling and blocked tissue reparative functions of macrophages in the regressing plaque, including M2-polarization, efferocytosis and sensing of specialized pro-resolving lipid mediators. Together, these data establish essential roles for Tregs in the resolution of atherosclerotic inflammation and plaque remodeling during regression.

Project description:Background：Atherosclerosis is the major cause of cardiovascular disease. Endothelial injury is one of the initiating factors of atherosclerosis. Injured endothelial cells overexpress adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), facilitating the binding and accumulation of monocytes-macrophages into the vascular wall via the ligands. Adipose-derived stem cells (ADSCs) have been extensively researched for their anti-inflammatory properties and therapeutic potential in various diseases. Targeting atherosclerotic regions with ADSCs may offer a promising therapeutic strategy to alleviate local inflammation associated with atherosclerosis.Methods：The ligands of very late antigen-4 (VLA-4) and lymphocyte function-associated antigen-1 (LFA-1) were expressed in the ADSC to construct the Macrophage (Mφ)-mimicking ADSCs. Cell proliferation, adhesion, and migration assays were used to characterize the Mφ-mimicking ADSCs. An atherosclerosis model was established in ApoE-/- mice via partial carotid artery ligation. Mφ-mimicking ADSCs were administered through tail vein injection. Distribution of these cells was tracked by far-red fluorescent probe. Histological analyses, including H&E staining, Oil Red O staining, and immunofluorescence were conducted to assess the impact on plaque size, composition, and macrophage polarization.Results：Mφ-mimicking ADSCs were successfully constructed and characterized. These cells exhibited enhanced adhesion and migration capabilities. The culture medium from Mφ-mimicking ADSCs increased the proportion of M2 macrophages and decreased the lipid uptake capacity. In the atherosclerosis mouse model, fluorescently labeled Mφ-mimicking ADSCs specifically adhered to and accumulated in atherosclerotic regions, demonstrating targeted localization. Mφ-mimicking ADSC treatment resulted in a reduction in plaque size and a decrease in the number of M1 macrophages within the arterial wall.Conclusions：Mφ-mimicking ADSCs confirmed their preserved anti-inflammatory properties and enhanced adhesion and migration abilities. In vivo, Mφ-mimicking ADSCs exhibited targeted localization ability to regions of endothelial dysfunction in atherosclerosis and attenuate plaque progression. These findings suggest that Mφ-mimicking ADSCs may serve as a potential therapeutic strategy for alleviating atherosclerosis and reducing inflammation.

Project description:Atherosclerosis is a chronic inflammatory disease of the blood vessels, characterized by atherosclerotic lesions. Early vascular injury initiates excessive inflammatory and immune responses, communicated by blood leukocytes and cells from the vasculature. Key factors contributing to early stages of atherosclerosis and plaque development include the pro-inflammatory cytokine IFNγ, which is vital for both innate and adaptive immunity and is also expressed at high levels in atherosclerotic lesions. Recent discoveries provide novel evidence to suggest that IFNγ-activated STAT1 orchestrates a platform of cell-type common and specific inflammatory responses in Vascular Smooth Muscle Cells (VSMC) and macrophages (MΦ) that are instrumental in the onset and progression of atherosclerotic plaque formation. Therefore, we compared genome-wide transcriptional responses of mouse VSMCs and bone marrow-derived macrophages (BMDMs) in time-dependent IFNγ response using RNA-seq. We identified genes upregulated both in a common and cell-type-specific manner. Next, comparative analysis with RNAseq data from aortic plaques from HFD-treated ApoEKO mice identified 225 differentially expressed genes with a BMDM-specific character. These included many known IFNγ target genes containing putative GAS and ISRE sites in their promoters. Likewise, using a scRNAseq data set obtained from human coronary atherosclerotic plaques, identified 98 pro-inflammatory and pro-atherogenic genes that were characterized as BMDM-specific and exclusively expressed in foamy, inflammatory, monocytes and tissue-resident BMDM subtypes. Finally, we identified a 25 BMDM-specific gene subset commonly expressed in mouse and human plaques, over-representing STAT1-binding sites and predominantly associated with Leukocyte Chemotaxis and Migration. These results provide strong evidence for a BMDM-specific role of STAT1 in coronary artery plaque development and identify a BMDM-specific STAT1-dependent pro-inflammatory gene signature that could serve to monitor and diagnose plaque progression in human atherosclerosis.

Project description:Background: Using proteomics, we strove to reveal novel molecular subtypes of human atherosclerotic lesions, study their associations with histology and imaging and relate them to long-term cardiovascular outcomes. Methods: 219 samples were obtained from 120 patients undergoing carotid endarterectomy. Sequential protein extraction was combined with multiplexed, discovery proteomics. Parallel reaction monitoring for 135 proteins was deployed for targeted validation. A combination of statistical, bioinformatics and machine learning methods was used to perform differential expression, network, pathway enrichment analysis and train and evaluate prognostic models. Results: Our extensive proteomics analysis from the core and periphery of plaques doubled the coverage of the plaque proteome compared to the largest proteomics study on atherosclerosis thus far. Plaque inflammation and calcification signatures were inversely correlated and validated with targeted proteomics. The inflammation signature was enriched with neutrophil-derived proteins, including calprotectin (S100A8/9) and myeloperoxidase. The calcification signature contained fetuin-A, osteopontin, and gamma-carboxylated proteins. Sex differences in the proteome of atherosclerosis were explained by a higher proportion of calcified plaques in women. Single-cell RNA sequencing data attributed the inflammation signature predominantly to neutrophils and macrophages and the calcification signature to smooth muscle cells, except for certain plasma proteins that were not expressed but retained in the plaque, i.e., fetuin-A. Echogenic lesions reflect the collagen content and calcification of plaque but carotid Duplex ultrasound fails to capture the extent of inflammatory protein changes in symptomatic plaques. Applying dimensionality reduction and machine learning on the proteomics data defined 4 distinct plaque phenotypes and revealed key protein signatures linked to smooth muscle cell content, plaque calcification and structural extracellular matrix, which improved the 9-year prognostic AUC by 25% compared to ultrasound and histology. A biosignature of four proteins (CNN1, PROC, SERPH, and CSPG2) independently predicted the progression of atherosclerosis and cardiovascular mortality with an AUC of 75% Conclusion: We combined discovery and targeted proteomics with network reconstruction and clustering techniques to provide molecular insights into protein changes in atherosclerotic plaques. The application of proteomics and machine learning techniques revealed distinct clusters of plaques that inform on disease progression and future adverse cardiovascular events.

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The role of macrophage VCAM-1 in atherosclerosis and inflammation

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