The MMP-9 promoter genetic variant rs3918242, mRNA and protein expression in advanced carotid plaque tissue.

BACKGROUND: The MMP-9 is a known player in atherosclerosis, yet associations of the MMP-9 -1562 C/T variant (rs3918242) with various atherosclerotic phenotypes and tissue mRNA expression are still contradictory. This study aimed to investigate the MMP-9 -1562 C/T variant, its mRNA and protein expression in carotid plaque (CP) tissue, as a risk factor for CP presence and as a marker of different plaque phenotypes (hyperechoic and hypoechoic) in patients undergoing carotid endarterectomy. The MnSOD as an MMP-9 negative regulator was also studied in relation to CP phenotypes. METHODS AND RESULTS: Genotyping of 770 participants (285 controls/485 patients) was done by tetra-primer ARMS PCR. The MMP-9 mRNA expression in 88 human CP tissues was detected by TaqMan® technology. The protein levels of MMP-9 and MnSOD were assessed by Western blot analysis. The MMP-9 -1562 C/T variant was not recognized as a risk factor for plaque presence or in predisposing MMP-9 mRNA and protein levels in plaque tissue. Patients with hypoechoic plaques had significantly lower MMP-9 mRNA and protein levels than those with hyperechoic plaque (p = 0.008, p = 0.003, respectively). MnSOD protein level was significantly higher in hypoechoic plaque compared to hyperechoic (p = 0.039). MMP-9 protein expression in CP tissue was significantly affected by sex and plaque type interaction (p = 0.009). CONCLUSIONS: Considering the differences of MMP-9 mRNA and protein expression in CP tissue regarding different plaque phenotypes and the observed sex-specific effect, the role of MMP-9 in human atherosclerotic plaques should be further elucidated.

RSAD2 is abundant in atherosclerotic plaques and promotes interferon-induced CXCR3-chemokines in human smooth muscle cells.

In atherosclerotic lesions, monocyte-derived macrophages are major source of interferon gamma (IFN-γ), a pleotropic cytokine known to regulate the expression of numerous genes, including the antiviral gene RSAD2. While RSAD2 was reported to be expressed in endothelial cells of human carotid lesions, its significance for the development of atherosclerosis remains utterly unknown. Here, we harnessed publicly available human carotid atherosclerotic data to explore RSAD2 in lesions and employed siRNA-mediated gene-knockdown to investigate its function in IFN-γ-stimulated human aortic smooth muscle cells (hAoSMCs). Silencing RSAD2 in IFN-γ-stimulated hAoSMCs resulted in reduced expression and secretion of key CXCR3-chemokines, CXCL9, CXCL10, and CXCL11. Conditioned medium from RSAD2-deficient hAoSMCs exhibited diminished monocyte attraction in vitro compared to conditioned medium from control cells. Furthermore, RSAD2 transcript was elevated in carotid lesions where it was expressed by several different cell types, including endothelial cells, macrophages and smooth muscle cells. Interestingly, RSAD2 displayed significant correlations with CXCL10 (r = 0.45, p = 0.010) and CXCL11 (r = 0.53, p = 0.002) in human carotid lesions. Combining our findings, we uncover a novel role for RSAD2 in hAoSMCs, which could potentially contribute to monocyte recruitment in the context of atherosclerosis.

An update on the cell-free DNA-derived methylome as a non-invasive biomarker for coronary artery disease.

Cardiovascular diseases are the foremost contributor to global mortality, presenting a complex etiology and an expanding array of risk factors. Coronary artery disease characterized by atherosclerotic plaque build-up in the coronary arteries, imposes significant mortality and financial burdens, especially in low- and middle-income nations. The pathogenesis of coronary artery disease involves a multifaceted interplay of genetic, environmental, and epigenetic factors. Epigenetic regulation contributes to the dynamic control of gene expression without altering the underlying DNA sequence. The mounting evidence that highlights the pivotal role of epigenetic regulation in coronary artery disease development and progression, offering potential avenues for the development of novel diagnostic biomarkers and therapeutic targets. Abnormal DNA methylation patterns are linked to the modulation of gene expression involved in crucial processes like lipid metabolism, inflammation, and vascular function in the context of coronary artery disease. Cell-free DNA has become invaluable in tumor biology as a liquid biopsy, while its applications in coronary artery disease are limited, but intriguing. Atherosclerotic plaque rupture causes myocardial infarction, by depriving heart muscles of oxygen, releasing cell-free DNA from dead cardiac cells, and providing a minimally invasive source to explore tissue-specific epigenetic alterations. We discussed the methodologies for studying the global methylome and hydroxy-methylome landscape, their advantages, and limitations. It explores methylome alterations in coronary artery disease, considering risk factors and their relevance in coronary artery disease genesis. The review also details the implications of MI-derived cell-free DNA for developing minimally invasive biomarkers and associated challenges.

Predicting the presence of coronary plaques featuring high-risk characteristics using polygenic risk scores and targeted proteomics in patients with suspected coronary artery disease.

BACKGROUND: The presence of coronary plaques with high-risk characteristics is strongly associated with adverse cardiac events beyond the identification of coronary stenosis. Testing by coronary computed tomography angiography (CCTA) enables the identification of high-risk plaques (HRP). Referral for CCTA is presently based on pre-test probability estimates including clinical risk factors (CRFs); however, proteomics and/or genetic information could potentially improve patient selection for CCTA and, hence, identification of HRP. We aimed to (1) identify proteomic and genetic features associated with HRP presence and (2) investigate the effect of combining CRFs, proteomics, and genetics to predict HRP presence. METHODS: Consecutive chest pain patients (n = 1462) undergoing CCTA to diagnose obstructive coronary artery disease (CAD) were included. Coronary plaques were assessed using a semi-automatic plaque analysis tool. Measurements of 368 circulating proteins were obtained with targeted Olink panels, and DNA genotyping was performed in all patients. Imputed genetic variants were used to compute a multi-trait multi-ancestry genome-wide polygenic score (GPSMult). HRP presence was defined as plaques with two or more high-risk characteristics (low attenuation, spotty calcification, positive remodeling, and napkin ring sign). Prediction of HRP presence was performed using the glmnet algorithm with repeated fivefold cross-validation, using CRFs, proteomics, and GPSMult as input features. RESULTS: HRPs were detected in 165 (11%) patients, and 15 input features were associated with HRP presence. Prediction of HRP presence based on CRFs yielded a mean area under the receiver operating curve (AUC) ± standard error of 73.2 ± 0.1, versus 69.0 ± 0.1 for proteomics and 60.1 ± 0.1 for GPSMult. Combining CRFs with GPSMult increased prediction accuracy (AUC 74.8 ± 0.1 (P = 0.004)), while the inclusion of proteomics provided no significant improvement to either the CRF (AUC 73.2 ± 0.1, P = 1.00) or the CRF + GPSMult (AUC 74.6 ± 0.1, P = 1.00) models, respectively. CONCLUSIONS: In patients with suspected CAD, incorporating genetic data with either clinical or proteomic data improves the prediction of high-risk plaque presence. TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT02264717 (September 2014).

Elevated Hepcidin Expression in Human Carotid Atheroma: Sex-Specific Differences and Associations with Plaque Vulnerability.

Hepcidin is upregulated by increased body iron stores and inflammatory cytokines. It is associated with cardiovascular events, arterial stiffness, and increased iron accumulation in human atheroma with hemorrhage. However, it is unknown whether the expression of hepcidin in human carotid plaques is related to plaque severity and whether hepcidin expression differs between men and women. Carotid samples from 58 patients (38 males and 20 females) were immunostained with hepcidin, macrophages, ferritin, and transferrin receptor. Immunocytochemistry of hepcidin was performed on THP-1 macrophages exposed to iron or 7betahydroxycholesterol. Hepcidin expression significantly increases with the progression of human atherosclerotic plaques. Plaques of male patients have significantly higher levels of hepcidin. Expressions of hepcidin are significantly correlated with the accumulation of CD68-positive macrophages and transferrin receptor 1 (TfR1) and apoptosis. In vitro, hepcidin is significantly increased in macrophages exposed to iron and moderately increased following 7-oxysterol treatment. In the cultured cells, suppression of hepcidin protected against macrophage cell death, lysosomal membrane permeabilization, and oxidative stress. Hepcidin may play a crucial role in the development and progression of atherosclerosis. The differential expression of hepcidin in male and female patients and its significant correlations with plaque severity, highlight the potential of hepcidin as a biomarker for risk stratification and therapeutic targeting in atherosclerosis.

[Zhuyu Pills promote polarization of macrophages toward M2 phenotype to prevent atherosclerosis via PPARγ/NF-κB signaling pathway].

This article aims to investigate the effect of Zhuyu Pills on atherosclerosis and decipher the underlying mechanism. The mouse model of atherosclerosis was induced by a high-fat diet, and the total modeling period was 12 weeks. A total of 47 ApoE~(-/-) mice successfully modeled were randomized into 5 groups, including 10 in the model group, 9 in each of low-, medium-, and high-dose(130.54, 261.08 and 522.16 mg·kg~(-1)·d~(-1), respectively) Zhuyu Pills groups, and 10 in the atorvastatin calcium(10.40 mg·kg~(-1)·d~(-1)) group. In addition, 10 C57BL/6J mice were included as the normal group. The mice in the normal group and model group were administrated with an equal volume of sterile distilled water, and those in other groups with corresponding agents by gavage once a day for 12 weeks. At the end of drug intervention, the levels of total cholesterol(TC), triglyceride(TG), high-density lipoprotein cholesterol(HDL-C), and low-density lipoprotein cholesterol(LDL-C) were measured by the biochemical method. Hematoxylin-eosin(HE) staining was employed to observe the plaque distribution in the aortic region. The serum levels of pro-inflammatory cytokines tumor necrosis factor-α(TNF-α) and interleukin(IL)-6 in M1 macrophages and anti-inflammatory cytokines IL-13 and IL-4 in M2 macrophages were determined by enzyme-linked immunosorbent assay(ELISA). The expression levels of inducible nitric oxide synthase(iNOS) and arginase-1(Arg-1) were examined by immunofluorescence. Real-time fluorescence quantitative polymerase chain reaction(real-time PCR) was employed to measure the mRNA levels of peroxisome proliferator-activated receptor γ(PPARγ), nuclear factor-κB(NF-κB), Arg-1, and iNOS in the aorta. Western blot was employed to determine the protein levels of PPARγ and NF-κB in the aorta. The results showed that compared with the normal group, the modeling elevated the TC, TG, and LDL-C levels, lowered the HDL-C level, caused large area thickening of the aortic intima, elevated the TNF-α and IL-6 levels, lowered the IL-4 and IL-13 levels, down-regulated the mRNA and protein levels of PPARγ and Arg-1, and up-regulated the mRNA and protein levels of iNOS and NF-κB in the aorta(P<0.01). Compared with the model group, low-, medium-, and high-dose Zhuyu Pills and atorvastatin calcium lowered the TC, TG, and LDL-C levels, elevated the HDL-C level, reduced the plaque area in a concentration-dependent manner, lowered the TNF-α and IL-6 levels, elevated the IL-4 and IL-13 levels, up-regulated the mRNA and protein levels of PPARγ and Arg-1, and down-regulated the mRNA and protein levels of NF-κB and iNOS in the aorta(P<0.05 or P<0.01). In conclusion, Zhuyu Pills may play an anti-atherosclerosis role by regulating PPARγ/NF-κB signaling pathway, inhibiting the polarization of macrophages toward the M1 phenotype, promoting the polarization of macrophages toward the M2 phenotype, and improving the inflammatory microenvironment of macrophages.

Transcription Factor Regulation of Gene Expression Network by ZNF385D and HAND2 in Carotid Atherosclerosis.

Carotid intima-media thickness (CIMT) is a surrogate indicator for atherosclerosis and has been shown to predict cardiovascular risk in multiple large studies. Identification of molecular markers for carotid atheroma plaque formation can be critical for early intervention and prevention of atherosclerosis. This study performed transcription factor (TF) network analysis of global gene expression data focusing on two TF genes, ZNF385D and HAND2, whose polymorphisms have been recently reported to show association with CIMT. Genome-wide gene expression data were measured from pieces of carotid endarterectomy collected from 34 hypertensive patients (atheroma plaque of stages IV and above according to the Stary classification) each paired with one sample of distant macroscopically intact tissue (stages I and II). Transcriptional regulation networks or the regulons were reconstructed for ZNF385D (5644 target genes) and HAND2 (781 target genes) using network inference. Their association with the progression of carotid atheroma was examined using gene-set enrichment analysis with extremely high statistical significance for regulons of both ZNF385D and HAND2 (p < 6.95 × 10-7) suggesting the involvement of expression quantitative loci (eQTL). Functional annotation of the regulon genes found heavy involvement in the immune system's response to inflammation and infection in the development of atherosclerosis. Detailed examination of the regulation and correlation patterns suggests that activities of the two TF genes could have high clinical and interventional impacts on impairing carotid atheroma plaque formation and preventing carotid atherosclerosis.

Re-analysis of single-cell transcriptomics reveals a critical role of macrophage-like smooth muscle cells in advanced atherosclerotic plaque.

Aims: Smooth muscle cell (SMC) remodeling poses a critical feature in the development and progression of atherosclerosis. Although fate mapping and in silicon approaches have expanded SMC phenotypes in atherosclerosis, it still remains elusive about the contributions of individual SMC phenotypes and molecular dynamics to advanced atherosclerotic plaque. Methods: Using single-cell transcriptome, we investigated cellular compositions of human carotid plaque laden with atherosclerotic core, followed by in vivo experiments utilizing SMC-lineage tracing technology, bulk RNA sequencing (RNA-seq) and both in vivo and in vitro validation of the underlying molecular mechanism. Results: 5 functionally distinct SMC subtypes were uncovered based on transcriptional features (described as contractile, fibroblast-like, osteogenic, synthetic and macrophage-like) within the niche. A proinflammatory, macrophage-like SMC subtype displaying an intermediary phenotype between SMC and macrophage, exhibits prominent potential in destabilizing plaque. At the molecular level, we explored cluster-specific master regulons by algorithm, and identified interferon regulatory factor-8 (IRF8) as a potential stimulator of SMC-to-macrophage transdifferentiation via activating nuclear factor-κB (NF-κB) signaling. Conclusions: Our study illustrates a comprehensive cell atlas and molecular landscape of advanced atherosclerotic lesion, which might renovate current understanding of SMC biology in atherosclerosis.

Comprehensive analysis identifies crucial genes associated with immune cells mediating progression of carotid atherosclerotic plaque.

BACKGROUNDS: Carotid atherosclerosis is prone to rupture and cause ischemic stroke in advanced stages of development. Our research aims to provide markers for the progression of atherosclerosis and potential targets for its treatment. METHODS: We performed a thorough analysis using various techniques including DEGs, GO/KEGG, xCell, WGCNA, GSEA, and other methods. The gene expression omnibus datasets GSE28829 and GSE43292 were utilized for this comprehensive analysis. The validation datasets employed in this study consisted of GSE41571 and GSE120521 datasets. Finally, we validated PLEK by immunohistochemistry staining in clinical samples. RESULTS: Using the WGCNA technique, we discovered 636 differentially expressed genes (DEGs) and obtained 12 co-expression modules. Additionally, we discovered two modules that were specifically associated with atherosclerotic plaque. A total of 330 genes that were both present in DEGs and WGCNA results were used to create a protein-protein network in Cytoscape. We used four different algorithms to get the top 10 genes and finally got 6 overlapped genes (TYROBP, ITGB2, ITGAM, PLEK, LCP2, CD86), which are identified by GSE41571 and GSE120521 datasets. Interestingly, the area under curves (AUC) of PLEK is 0.833. Besides, we found PLEK is strongly positively correlated with most lymphocytes and myeloid cells, especially monocytes and macrophages, and negatively correlated with most stromal cells (e.g, neurons, myocytes, and fibroblasts). The expression of PLEK were consistent with the immunohistochemistry results. CONCLUSIONS: Six genes (TYROBP, ITGB2, ITGAM, PLEK, LCP2, CD86) were found to be connected with carotid atherosclerotic plaques and PLEK may be an important biomarker and a potential therapeutic target.

Comprehensive Analysis of 1-Year-Old Female Apolipoprotein E-Deficient Mice Reveals Advanced Atherosclerosis with Vulnerable Plaque Characteristics.

Apolipoprotein E-knockout (Apoe-/-) mice constitute the most widely employed animal model of atherosclerosis. Deletion of Apoe induces profound hypercholesterolemia and promotes the development of atherosclerosis. However, despite its widespread use, the Apoe-/- mouse model remains incompletely characterized, especially at late time points and advanced disease stages. Thus, it is unclear how late atherosclerotic plaques compare to earlier ones in terms of lipid deposition, calcification, macrophage accumulation, smooth muscle cell presence, or plaque necrosis. Additionally, it is unknown how cardiac function and hemodynamic parameters are affected at late disease stages. Here, we used a comprehensive analysis based on histology, fluorescence microscopy, and Doppler ultrasonography to show that in normal chow diet-fed Apoe-/- mice, atherosclerotic lesions at the level of the aortic valve evolve from a more cellular macrophage-rich phenotype at 26 weeks to an acellular, lipid-rich, and more necrotic phenotype at 52 weeks of age, also marked by enhanced lipid deposition and calcification. Coronary artery atherosclerotic lesions are sparse at 26 weeks but ubiquitous and extensive at 52 weeks; yet, left ventricular function was not significantly affected. These findings demonstrate that atherosclerosis in Apoe-/- mice is a highly dynamic process, with atherosclerotic plaques evolving over time. At late disease stages, histopathological characteristics of increased plaque vulnerability predominate in combination with frequent and extensive coronary artery lesions, which nevertheless may not necessarily result in impaired cardiac function.

Identification of key pyroptosis-related genes and microimmune environment among peripheral arterial beds in atherosclerotic arteries.

Atherosclerosis is a chronic inflammatory disease characterized with innate and adaptive immunity but also involves pyroptosis. Few studies have explored the role of pyroptosis in advanced atherosclerotic plaques from different vascular beds. Here we try to identify the different underlying function of pyroptosis in the progression of atherosclerosis between carotid arteries and femoral. arteries. We extracted gene expression levels from 55 advanced carotid or femoral atherosclerotic plaques. The pyroptosis score of each sample was calculated by single-sample-gene-set enrichment analysis (ssGSEA). We then divided the samples into two clusters: high pyroptosis scores cluster (PyroptosisScoreH cluster) and low pyroptosis scores cluster (PyroptosisScoreL cluster), and assessed functional enrichment and immune cell infiltration in the two clusters. Key pyroptosis related genes were identified by the intersection between results of Cytoscape and LASSO (Least Absolute Shrinkage and Selection Operator) regression analysis. Finally, all key pyroptosis related genes were validated in vitro. We found all but one of the 29 carotid plaque samples belonged to the PyroptosisScoreH cluster and the majority (19 out of 26) of femoral plaques were part of the PyroptosisScoreL cluster. Atheromatous plaque samples in the PyroptosisScoreL cluster had higher proportions of gamma delta T cells, M2 macrophages, myeloid dendritic cells (DCs), and cytotoxic lymphocytes (CTLs), but lower proportions of endothelial cells (ECs). Immune full-activation pathways (e.g., NOD-like receptor signaling pathway and NF-kappa B signaling pathway) were highly enriched in the PyroptosisScoreH cluster. The key pyroptosis related genes GSDMD, CASP1, NLRC4, AIM2, and IL18 were upregulated in advanced carotid atherosclerotic plaques. We concluded that compared to advanced femoral atheromatous plaques, advanced carotid atheromatous plaques were of higher grade of pyroptosis. GSDMD, CASP1, NLRC4, AIM2, and IL18 were the key pyroptosis related genes, which might provide a new sight in the prevention of fatal strokes in advanced carotid atherosclerosis.

Omics Approaches Unveiling the Biology of Human Atherosclerotic Plaques.

Atherosclerosis is a chronic inflammatory disease of the arterial wall, characterized by the buildup of plaques with the accumulation and transformation of lipids, immune cells, vascular smooth muscle cells, and necrotic cell debris. Plaques with collagen-poor thin fibrous caps infiltrated by macrophages and lymphocytes are considered unstable because they are at the greatest risk of rupture and clinical events. However, the current histologic definition of plaque types may not fully capture the complex molecular nature of atherosclerotic plaque biology and the underlying mechanisms contributing to plaque progression, rupture, and erosion. The advances in omics technologies have changed the understanding of atherosclerosis plaque biology, offering new possibilities to improve risk prediction and discover novel therapeutic targets. Genomic studies have shed light on the genetic predisposition to atherosclerosis, and integrative genomic analyses expedite the translation of genomic discoveries. Transcriptomic, proteomic, metabolomic, and lipidomic studies have refined the understanding of the molecular signature of atherosclerotic plaques, aiding in data-driven hypothesis generation for mechanistic studies and offering new prospects for biomarker discovery. Furthermore, advancements in single-cell technologies and emerging spatial analysis techniques have unveiled the heterogeneity and plasticity of plaque cells. This review discusses key omics-based discoveries that have advanced the understanding of human atherosclerotic plaque biology, focusing on insights derived from omics profiling of human atherosclerotic vascular specimens.

Identification of a hub gene VCL for atherosclerotic plaques and discovery of potential therapeutic targets by molecular docking.

BACKGROUND: Atherosclerosis (AS) is a pathology factor for cardiovascular diseases and instability of atherosclerotic plaques contributes to acute coronary events. This study identified a hub gene VCL for atherosclerotic plaques and discovered its potential therapeutic targets for atherosclerotic plaques. METHODS: Differential expressed genes (DEGs) were screened between unstable and stable plaques from GSE120521 dataset and then used for construction of a protein-protein interactions (PPI) network. Through topological analysis, hub genes were identified within this PPI network, followed by construction of a diagnostic model. GSE41571 dataset was utilized to validate the diagnostic model. A key hub gene was identified and its association with immune characteristics and pathways were further investigated. Molecular docking and molecular dynamics (MD) simulation were employed to discover potential therapeutic targets. RESULTS: According to the PPI network, 3 tightly connected protein clusters were found. Topological analysis identified the top 5 hub genes, Vinculin (VCL), Dystrophin (DMD), Actin alpha 2 (ACTA2), Filamin A (FLNA), and transgelin (TAGLN). Among these hub genes, VCL had the highest diagnostic value. VCL was selected for further analysis and we found that VCL was negatively correlated with immune score and AS-related inflammatory pathways. Next, we identified 408 genes that were highly correlated with VCL and determined potential drug candidates. The results from molecular docking and MD simulation showed compound DB07117 combined with VCL protein stably, the binding energy is -7.7 kcal/mol, indicating that compound DB07117 was a potential inhibitor of VCL protein. CONCLUSION: This study identified VCL as a key gene for atherosclerotic plaques and provides a potential therapeutic target of VCL for the treatment of atherosclerotic plaques.

Unveiling the role of ABI3 and hub senescence-related genes in macrophage senescence for atherosclerotic plaque progression.

BACKGROUND: Atherosclerosis, characterized by abnormal arterial lipid deposition, is an age-dependent inflammatory disease and contributes to elevated morbidity and mortality. Senescent foamy macrophages are considered to be deleterious at all stages of atherosclerosis, while the underlying mechanisms remain largely unknown. In this study, we aimed to explore the senescence-related genes in macrophages diagnosis for atherosclerotic plaque progression. METHODS: The atherosclerosis-related datasets were retrieved from the Gene Expression Omnibus (GEO) database, and cellular senescence-associated genes were acquired from the CellAge database. R package Limma was used to screen out the differentially expressed senescence-related genes (DE-SRGs), and then three machine learning algorithms were applied to determine the hub DE-SRGs. Next, we established a nomogram model to further confirm the clinical significance of hub DE-SRGs. Finally, we validated the expression of hub SRG ABI3 by Sc-RNA seq analysis and explored the underlying mechanism of ABI3 in THP-1-derived macrophages and mouse atherosclerotic lesions. RESULTS: A total of 15 DE-SRGs were identified in macrophage-rich plaques, with five hub DE-SRGs (ABI3, CAV1, NINJ1, Nox4 and YAP1) were further screened using three machine learning algorithms. Subsequently, a nomogram predictive model confirmed the high validity of the five hub DE-SRGs for evaluating atherosclerotic plaque progression. Further, the ABI3 expression was upregulated in macrophages of advanced plaques and senescent THP-1-derived macrophages, which was consistent with the bioinformatics analysis. ABI3 knockdown abolished macrophage senescence, and the NF-κB signaling pathway contributed to ABI3-mediated macrophage senescence. CONCLUSION: We identified five cellular senescence-associated genes for atherogenesis progression and unveiled that ABI3 might promote macrophage senescence via activation of the NF-κB pathway in atherogenesis progression, which proposes new preventive and therapeutic strategies of senolytic agents for atherosclerosis.

The Characteristics of Macrophage Heterogeneity in Atherosclerotic Aortas.

Macrophage is the main effector cell during atherosclerosis. We applied single-cell RNA sequencing (scRNA) data to investigate the role of macrophage subsets in atherosclerosis. Monocyte and macrophage clusters were divided into 6 subclusters. Each subcluster's markers were calculated and validated by immunofluorescence. Elevated macrophage subclusters in the WD group were subject to enrichment pathway analysis and exhibited different phenotypes. Pseudotime analysis shows the subclusters originate from monocytes. We cultured bone marrow-derived macrophages with CSF-1 and ox-LDL to simulate an atherosclerotic-like environment and detected the transformation of subclusters. Macrophage-Vegfa and Macrophage-C1qb increased in the WD group. Macrophage-Vegfa acquires the characteristics of phagocytosis and immune response, while Macrophage-C1qb is not involved in lipid metabolism. The two subclusters are both enriched in cell movement and migration pathways. Experimental verification proved Monocyte-Ly6C evolved into Macrophage-Vegfa and Macrophage-C1qb during atherosclerosis progression.

Identification of co-expressed genes and immune infiltration features related to the progression of atherosclerosis.

Atherosclerosis is a chronic inflammatory disease that affects arterial walls and is a leading cause of cardiovascular disease. Gene co-expression modules can provide insight into the molecular mechanisms underlying atherosclerosis progression. In this study, gene co-expression network analysis (WGCNA) was done to identify gene co-expression modules associated with atherosclerosis progression. Before conducting WGCNA, preprocessing and soft power selection were performed on the GSE28829, GSE100927, GSE43292, GSE10334, and GSE16134 datasets ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi ). Co-expression modules were identified using dynamic tree cuts, and their correlations and trait associations were visualized. Enrichment analysis was performed on the blue and magenta modules to identify biological processes (BP) and pathways related to atherosclerosis. The CIBERSORT algorithm was used to predict immune cell infiltration in early and advanced atherosclerotic plaques. We identified 12 co-expression modules, in which blue and magenta were most highly correlated with atherosclerosis progression. The blue module was enriched for inflammation- and immune-related BP and pathways, including phagosome, lysosome, osteoclast differentiation, chemokine signaling pathway, platelet activation, NF-kappa B signaling pathway, Fc gamma R-mediated phagocytosis, lipid and atherosclerosis, autophagy, and apoptosis. The magenta module was significantly enriched for vascular permeability regulation, positive and negative regulation of epithelial to mesenchymal transition, and lamellipodium. Additionally, the CIBERSORT algorithm predicted less abundance of T regulatory cells and monocytes in advanced compared to early atherosclerotic plaques. The enrichment analysis of BP, cellular components, molecular functions, and atherosclerosis-related pathways in the blue and magenta modules showed that inflammation and immune response played a key role in the progression of atherosclerosis. Our study provides insights into the molecular mechanisms underlying atherosclerosis progression and identifies potential therapeutic targets for the treatment of atherosclerosis. The identification of immune cell subtypes associated with atherosclerosis could lead to the development of immunomodulatory therapies to prevent or treat atherosclerosis.

Developmental heterogeneity of vascular cells: Insights into cellular plasticity in atherosclerosis?

Smooth muscle cells, endothelial cells and macrophages display remarkable heterogeneity within the healthy vasculature and under pathological conditions. During development, these cells arise from numerous embryological origins, which confound with different microenvironments to generate postnatal vascular cell diversity. In the atherosclerotic plaque milieu, all these cell types exhibit astonishing plasticity, generating a variety of plaque burdening or plaque stabilizing phenotypes. And yet how developmental origin influences intraplaque cell plasticity remains largely unexplored despite evidence suggesting this may be the case. Uncovering the diversity and plasticity of vascular cells is being revolutionized by unbiased single cell whole transcriptome analysis techniques that will likely continue to pave the way for therapeutic research. Cellular plasticity is only just emerging as a target for future therapeutics, and uncovering how intraplaque plasticity differs across vascular beds may provide key insights into why different plaques behave differently and may confer different risks of subsequent cardiovascular events.

Influence of ferroptosis indicators on the stability of atherosclerotic plaque.

Ferroptosis is a new form of cell death that is unique and closely related to iron concentration, and reactive oxygen species (ROS) production. We investigated the indicators of ferroptosis between vulnerable plaque and stable plaque in atherosclerotic. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the expression of the ferroptosis-related genes and proteins and extracellular matrix stability-related genes and proteins (FN, CoL-1). Superoxide dismutase (SOD) activities, glutathione peroxidase (GSH) and malondialdehyde (MDA) were detected by ELISA. The commercially available kit was used to detect Fe2+ concentration in tissue. DCFH-DA fluorescent probe was used to detect the ROS levels. H&E stain, Masson trichrome stain, and Oil Red O stain were used to detect pathological states in vulnerable plaque and stable plaque. Tissue localization and positive rate of GPX4, SLC7A11, COX-2, FN, and COL-1 were evaluated by immunohistochemistry. The results showed a significant increase in the expression of COX2 and a significant decrease in the expression of GPX4 and SLC7A11 in genes related to ferroptosis in vulnerable plaque compared with stable plaque. Pathologic results showed vulnerable plaque with higher levels of inflammatory cell infiltration, more diffuse collagen fibers, and larger particles of lipid droplets. Concentrations of the antioxidant metabolites SOD and GSH were significantly reduced and concentrations of the oxidative metabolites MDA and Fe2+ were significantly increased in vulnerable plaque compared with stable plaque. The expression of FN and CoL-1 was significantly reduced in genes related to extracellular matrix stability in vulnerable plaque. Taken together, these findings indicate that the degree of ferroptosis in vulnerable plaque is higher than that in stable plaque, suggesting that changes in indicators of ferroptosis may affect carotid atherosclerotic plaque stability, target spot in the ferroptosis signaling pathway may provide further theoretical basis for the clinical treatment of carotid atherosclerosis.

DNA methylation and histone post-translational modifications in atherosclerosis and a novel perspective for epigenetic therapy.

Atherosclerosis, which is a vascular pathology characterized by inflammation and plaque build-up within arterial vessel walls, acts as the important cause of most cardiovascular diseases. Except for a lipid-depository and chronic inflammatory, increasing evidences propose that epigenetic modifications are increasingly associated with atherosclerosis and are of interest from both therapeutic and biomarker perspectives. The chronic progressive nature of atherosclerosis has highlighted atherosclerosis heterogeneity and the fact that specific cell types in the complex milieu of the plaque are, by far, not the only initiators and drivers of atherosclerosis. Instead, the ubiquitous effects of cell type are tightly controlled and directed by the epigenetic signature, which, in turn, is affected by many proatherogenic stimuli, including low-density lipoprotein, proinflammatory, and physical forces of blood circulation. In this review, we summarize the role of DNA methylation and histone post-translational modifications in atherosclerosis. The future research directions and potential therapy for the management of atherosclerosis are also discussed. Video Abstract.

LITAF Promotes Atherosclerotic Plaque Formation by Stimulating the NF-κB Inflammatory Pathway.

OBJECTIVE: Lipopolysaccharide-induced tumor necrosis factor-α factor (LITAF) protein is a newly discovered inflammatory protein. This study aims to study the role of LITAF in the formation of atherosclerosis. METHODS: A total of 10 C57BL/6J mice and 10 C57BL/6J mice with knockout of LITAF gene (C57BL/6J-LITAF-) were divided into two groups: the control group and the LITAF-/- group. The animals were accommodated for 16 weeks and then euthanized with their hearts and aortas isolated thereafter. Next, the roots of the mouse aorta were cryosectioned and stained with Oil Red O staining and immunohistochemical staining (CD68, α-SMA, and Masson), respectively. The area of Oil Red O staining and the proportion of positive expression after immunohistochemical staining were then compared between the control and LITAF-/- groups. At the same time, the blood of mice was collected for the extraction of proteins and RNA. The proteins and RNA were used to detect the expression of major molecules of the NF-κB inflammatory pathway in mice in the control group and the LITAF-/- group by Western blotting and RT-PCR. RESULTS: Oil Red O staining of the aortic root sections of the mice in each group revealed that the area of atherosclerotic plaques in the LITAF-/- group was substantially lower than that in the control group (P<0.05). Moreover, immunohistochemical staining determined that the expression level of α-SMA and CD68 in the LITAF-/- group was significantly lower than that in the control group, whereas the results were reversed following Masson staining (P<0.05). The expression levels of P65 and caspase 3 were significantly lower in the LITAF-/- group than in the control group (P<0.05), whereas the expression level of IκB was higher in the LITAF-/- group. CONCLUSION: LITAF might participate in the formation of atherosclerotic plaque through the NF-κB pathway and play a promoting role in the formation of atherosclerosis.