Immune and inflammatory insights in atherosclerosis: development of a risk prediction model through single-cell and bulk transcriptomic analyses.

Background: Investigation into the immune heterogeneity linked with atherosclerosis remains understudied. This knowledge gap hinders the creation of a robust theoretical framework essential for devising personalized immunotherapies aimed at combating this disease. Methods: Single-cell RNA sequencing (scRNA-seq) analysis was employed to delineate the immune cell-type landscape within atherosclerotic plaques, followed by assessments of cell-cell interactions and phenotype characteristics using scRNA-seq datasets. Subsequently, pseudotime trajectory analysis was utilized to elucidate the heterogeneity in cell fate and differentiation among macrophages. Through integrated approaches, including single-cell sequencing, Weighted Gene Co-expression Network Analysis (WGCNA), and machine learning techniques, we identified hallmark genes. A risk score model and a corresponding nomogram were developed and validated using these genes, confirmed through Receiver Operating Characteristic (ROC) curve analysis. Additionally, enrichment and immune characteristic analyses were conducted based on the risk score model. The model's applicability was further corroborated by in vitro and in vivo validation of specific genes implicated in atherosclerosis. Result: This comprehensive scRNA-seq analysis has shed new light on the intricate immune landscape and the role of macrophages in atherosclerotic plaques. The presence of diverse immune cell populations, with a particularly enriched macrophage population, was highlighted by the results. Macrophage heterogeneity was intricately characterized, revealing four distinct subtypes with varying functional attributes that underscore their complex roles in atherosclerotic pathology. Intercellular communication analysis revealed robust macrophage interactions with multiple cell types and detailed pathways differing between proximal adjacent and atherosclerotic core groups. Furthermore, pseudotime trajectories charted the developmental course of macrophage subpopulations, offering insights into their differentiation fates within the plaque microenvironment. The use of machine learning identified potential diagnostic markers, culminating in the identification of RNASE1 and CD14. The risk score model based on these biomarkers exhibited high accuracy in diagnosing atherosclerosis. Immune characteristic analysis validated the risk score model's efficacy in defining patient profiles, distinguishing high-risk individuals with pronounced immune cell activities. Finally, experimental validation affirmed RNASE1's involvement in atherosclerotic progression, suggesting its potential as a therapeutic target. Conclusion: Our findings have advanced our understanding of atherosclerosis immunopathology and paved the way for novel diagnostic and therapeutic strategies.

Decoding marker genes and immune landscape of unstable carotid plaques from cellular senescence.

Recently, cellular senescence-induced unstable carotid plaques have gained increasing attention. In this study, we utilized bioinformatics and machine learning methods to investigate the correlation between cellular senescence and the pathological mechanisms of unstable carotid plaques. Our aim was to elucidate the causes of unstable carotid plaque progression and identify new therapeutic strategies. First, differential expression analysis was performed on the test set GSE43292 to identify differentially expressed genes (DEGs) between the unstable plaque group and the control group. These DEGs were intersected with cellular senescence-associated genes to obtain 40 cellular senescence-associated DEGs. Subsequently, key genes were then identified through weighted gene co-expression network analysis, random forest, Recursive Feature Elimination for Support Vector Machines algorithm and cytoHubba plugin. The intersection yielded 3 CSA-signature genes, which were validated in the external validation set GSE163154. Additionally, we assessed the relationship between these CSA-signature genes and the immune landscape of the unstable plaque group. This study suggests that cellular senescence may play an important role in the progression mechanism of unstable plaques and is closely related to the influence of the immune microenvironment. Our research lays the foundation for studying the progression mechanism of unstable carotid plaques and provides some reference for targeted therapy.

Knock-Out of IKKepsilon Ameliorates Atherosclerosis and Fatty Liver Disease by Alterations of Lipid Metabolism in the PCSK9 Model in Mice.

The inhibitor-kappaB kinase epsilon (IKKε) represents a non-canonical IκB kinase that modulates NF-κB activity and interferon I responses. Inhibition of this pathway has been linked with atherosclerosis and metabolic dysfunction-associated steatotic liver disease (MASLD), yet the results are contradictory. In this study, we employed a combined model of hepatic PCSK9D377Y overexpression and a high-fat diet for 16 weeks to induce atherosclerosis and liver steatosis. The development of atherosclerotic plaques, serum lipid concentrations, and lipid metabolism in the liver and adipose tissue were compared between wild-type and IKKε knock-out mice. The formation and progression of plaques were markedly reduced in IKKε knockout mice, accompanied by reduced serum cholesterol levels, fat deposition, and macrophage infiltration within the plaque. Additionally, the development of a fatty liver was diminished in these mice, which may be attributed to decreased levels of multiple lipid species, particularly monounsaturated fatty acids, triglycerides, and ceramides in the serum. The modulation of several proteins within the liver and adipose tissue suggests that de novo lipogenesis and the inflammatory response are suppressed as a consequence of IKKε inhibition. In conclusion, our data suggest that the knockout of IKKε is involved in mechanisms of both atherosclerosis and MASLD. Inhibition of this pathway may therefore represent a novel approach to the treatment of cardiovascular and metabolic diseases.

MicroRNA Inhibiting Atheroprotective Proteins in Patients with Unstable Angina Comparing to Chronic Coronary Syndrome.

Patients with unstable angina present clinical characteristics of atherosclerotic plaque vulnerability, contrary to chronic coronary syndrome patients. The process of athersclerotic plaque destabilization is also regulated by microRNA particles. In this study, the investigation on expression levels of microRNAs inhibiting the expression of proteins that protect from atherosclerotic plaque progression (miR-92a inhibiting KLF2, miR-10b inhibiting KLF4, miR-126 inhibiting MerTK, miR-98 inhibiting IL-10, miR-29b inhibiting TGFß1) was undertaken. A number of 62 individuals were enrolled-unstable angina (UA, n = 14), chronic coronary syndrome (CCS, n = 38), and healthy volunteers (HV, n = 10). Plasma samples were taken, and microRNAs expression levels were assessed by qRT-PCR. As a result, the UA patients presented significantly increased miR-10b levels compared to CCS patients (0.097 vs. 0.058, p = 0.033). Moreover, in additional analysis when UA patients were grouped together with stable patients with significant plaque in left main or proximal left anterior descending ("UA and LM/proxLAD" group, n = 29 patients) and compared to CCS patients with atherosclerotic lesions in other regions of coronary circulation ("CCS other" group, n = 25 patients) the expression levels of both miR-10b (0.104 vs. 0.046; p = 0.0032) and miR-92a (92.64 vs. 54.74; p = 0.0129) were significantly elevated. In conclusion, the study revealed significantly increased expression levels of miR-10b and miR-92a, a regulator of endothelial protective KLF factors (KLF4 and KLF2, respectively) in patients with more vulnerable plaque phenotypes.

High expression levels of haem oxygenase-1 promote ferroptosis in macrophage-derived foam cells and exacerbate plaque instability.

Plaque rupture with consequent thrombosis is the leading cause of acute cardiovascular events, during which macrophage death is a hallmark. Ferroptosis is a pivotal intermediate link between early and advanced atherosclerosis. Existing evidence indicates the involvement of macrophage ferroptosis in plaque vulnerability; however, the exact mechanism remains elusive. The aim of this study was to explore key ferroptosis-related genes (FRGs) involved in plaque progression and the underlying molecular mechanisms involved. The expression landscape of FRGs was obtained from atherosclerosis-related GEO datasets. Molecular mechanism studies of ferroptosis were performed using bone marrow-derived macrophages (BMDMs) and macrophage-derived foam cells (MDFCs). Bioinformatics analysis and immunohistochemistry revealed that macrophage haem oxygenase-1 (HMOX1) is the key FRG involved in plaque destabilization. Hypoxic conditions induced a significant increase in Hmox1 expression in MDFCs but not in macrophages. In addition, the beneficial or deleterious effects of Hmox1 were dependent on the degree of Hmox1 induction. Hmox1 overexpression drove inflammatory responses and ferroptotic oxidative stress in MDFCs and aggravated the plaque burden in atherosclerotic model mice. Further mechanistic investigations demonstrated that hypoxia-mediated degradation of egl-9 family hypoxia-inducible factor 3 (Egln3) stabilized Hif1a, which subsequently promoted Hmox1 transcription. Our findings suggest that high Hmox1 expression under hypoxia is deleterious to MDFC viability and plaque stability, providing a reference for the management of acute cardiovascular events.

Early intermittent hyperlipidaemia alters tissue macrophages to fuel atherosclerosis.

Hyperlipidaemia is a major risk factor of atherosclerotic cardiovascular disease (ASCVD). Risk of cardiovascular events depends on cumulative lifetime exposure to low-density lipoprotein cholesterol (LDL-C) and, independently, on the time course of exposure to LDL-C, with early exposure being associated with a higher risk1. Furthermore, LDL-C fluctuations are associated with ASCVD outcomes2-4. However, the precise mechanisms behind this increased ASCVD risk are not understood. Here we find that early intermittent feeding of mice on a high-cholesterol Western-type diet (WD) accelerates atherosclerosis compared with late continuous exposure to the WD, despite similar cumulative circulating LDL-C levels. We find that early intermittent hyperlipidaemia alters the number and homeostatic phenotype of resident-like arterial macrophages. Macrophage genes with altered expression are enriched for genes linked to human ASCVD in genome-wide association studies. We show that LYVE1+ resident macrophages are atheroprotective, and identify biological pathways related to actin filament organization, of which alteration accelerates atherosclerosis. Using the Young Finns Study, we show that exposure to cholesterol early in life is significantly associated with the incidence and size of carotid atherosclerotic plaques in mid-adulthood. In summary, our results identify early intermittent exposure to cholesterol as a strong determinant of accelerated atherosclerosis, highlighting the importance of optimal control of hyperlipidaemia early in life, and providing insights into the underlying biological mechanisms. This knowledge will be essential to designing effective therapeutic strategies to combat ASCVD.

Rare RNF213 variants is related to early-onset intracranial atherosclerosis: A Chinese community-based study.

BACKGROUND: The relationship between rare variants in Ring finger protein 213 (RNF213) and intracranial atherosclerosis (ICAS) remained unelucidated. Using whole-exome sequencing (WES) and high-resolution magnetic resonance imaging (HR-MRI), this study aimed at investigating the association between rare RNF213 variants and ICAS within a Chinese community-dwelling population. METHODS: The present study included 821 participants from Shunyi cohort. Genetic data of rare RNF213 variants were acquired by WES and were categorized by functional domains. Intracranial and extracranial atherosclerosis were assessed by brain HR-MRI and carotid ultrasound, respectively. Logistic regression and generalized linear regression were applied to evaluate the effects of rare RNF213 variants on atherosclerosis. Stratification by age were conducted with 50 years old set as the cutoff value. RESULTS: Ninety-five participants were identified as carriers of rare RNF213 variants. Carotid plaques were observed in 367 (44.7 %) participants, while ICAS was identified in 306 (37.3 %). Rare variants of RNF213 was not associated with ECAS. Employing HR-MRI, both the presence of rare variants (ß = 0.150, P = 0.025) and numerical count of variants (ß = 0.182, P = 0.003) were significantly correlated with ICAS within the group of age ≤50 years. Both variant existence (ß = 0.154, P = 0.014) and variant count (ß = 0.188, P = 0.003) were significantly associated with plaques in middle cerebral arteries within younger subgroup, rather than basilar arteries. Furthermore, a significant association was observed between variants that located outside the N-arm domain and ICAS in the younger subgroup (OR = 2.522, P = 0.030). Statistical results remained robust after adjusted for age, gender, and cardiovascular risk factors. CONCLUSIONS: Rare variants of RNF213 is associated with age-related ICAS in general Chinese population, highlighting the potential role of RNF213 as a genetic contributor to early-onset ICAS.

A cross-sectional study comparing the expression of DNA repair molecules in subjects with and without atherosclerotic plaques.

BACKGROUND: Atherosclerosis, serving as the primary pathological mechanism at the core of cardiovascular disease, is now widely acknowledged to be associated with DNA damage and repair, contributing to atherosclerotic plaque formation. Therefore, molecules involved in the DNA repair process may play an important role in the progression of atherosclerosis. Our research endeavors to explore the contributions of specific and interrelated molecules involved in DNA repair (APE1, BRCA1, ERCC2, miR-221-3p, miR-145-5p, and miR-155-5p) to the development of atherosclerotic plaque and their interactions with each other. METHODS & RESULTS: Gene expression study was conducted using the real-time polymerase chain reaction (qRT-PCR) method on samples from carotid artery atherosclerotic plaques and nonatherosclerotic internal mammary arteries obtained from 50 patients diagnosed with coronary artery disease and carotid artery disease. Additionally, 50 healthy controls were included for the determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Although no difference was observed in mRNA gene expressions, we noted a decrease in miR-155-5p gene expression (p = 0.003) and an increase in miR-221-3p gene expression (p = 0.015) in plaque samples, while miR-145-5p gene expression remained unchanged (p = 0.57). Regarding serum 8-OHdG levels, patients exhibited significantly higher levels (1111.82 ± 28.64) compared to controls (636.23 ± 24.23) (p < 0.0001). CONCLUSIONS: In our study demonstrating the role of miR-155-5p and miR-221-3p in atherosclerosis, we propose that these molecules are potential biomarkers and therapeutic targets for coronary artery diseases and carotid artery disease.

IRF9 and STAT1 as biomarkers involved in T-cell immunity in atherosclerosis.

Atherosclerosis is a common cardiovascular disease in which the arteries are thickened due to buildup of plaque. This study aims to identify programmed cell death (PCD)-related biomarkers and explore the crucial regulatory mechanisms of atherosclerosis. Gene expression profiles of atherosclerosis and control groups from GSE20129 and GSE23746 were obtained. Necroptosis was elevated in atherosclerosis. Weighted gene coexpression network analysis (WGCNA) was conducted in GSE23746 and GSE56045 to identify PCD-related modules and to perform enrichment analysis. Two necroptosis-related genes (IRF9 and STAT1) were identified and considered as biomarkers. Enrichment analysis showed that these gene modules were mainly related to immune response regulation. In addition, single-cell RNA sequencing data from GSE159677 were obtained and the characteristic cell types of atherosclerosis were identified. A total of 11 immune cell types were identified through UMAP dimension reduction. Most immune cells were mainly enriched in plaque samples, and STAT1 and IRF9 were primarily expressed in T-cells and macrophages. Moreover, the roles of IRF9 and STAT1 were assessed and found to be significantly upregulated in atherosclerosis, which was associated with increased risk of atherosclerosis. This study provides a molecular feature of atherosclerosis, offering an important basis for further research on its pathological mechanisms and the search for new therapeutic targets.

CTRP9: An Anti-Atherosclerotic Factor in ApoE Knockout Mice through Oxidative Stress Inhibition.

BACKGROUND: C1q/tumor necrosis factor-related protein-9 (CTRP9) is critically involved in the pathophysiology of metabolic and cardiovascular disorders. This investigation aimed to clarify the mechanism underlying the role of CTRP9 in atherosclerosis in apolipoprotein E (ApoE) knockout (KO) mice. METHODS: ApoE KO mice were fed a Western diet and injected with a virus which resulted in CTRP9 overexpression or knockdown for 12 weeks. The plasma lipid levels and atherosclerotic plaque areas were measured after the mice were euthanized. Aortas were isolated, and RNA sequencing was performed to identify the differentially expressed genes and related signaling pathways. Finally, plasma oxidative stress factors were measured to demonstrate the reliability of the RNA sequencing results. RESULTS: The plasma lipid levels in the CTRP9 overexpression group did not significantly differ from those in the green fluorescence protein (GFP) group. Markablely, CTRP9 overexpression inhibited atherosclerotic plaque formation in ApoE KO mice, whereas CTRP9 knockdown promoted plaque formation. RNA sequencing analysis identified 3485 differentially expressed genes that were prominently enriched across 55 signaling pathways. Additionally, plasma oxidative stress factors were significantly reduced after CTRP9 overexpression, whereas these factors were increased after CTRP9 knockdown, which was consistent with the results of the RNA sequencing analysis. CONCLUSIONS: These findings demonstrated that CTRP9 alleviated inflammation and cholesterol metabolism, which reduced oxidative stress in an atherosclerotic animal model. These beneficial effects may mediate the suppression of lesion development in the aorta.

Endothelial γ-protocadherins inhibit KLF2 and KLF4 to promote atherosclerosis.

Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of mortality worldwide. Laminar shear stress from blood flow, sensed by vascular endothelial cells, protects from ASCVD by upregulating the transcription factors KLF2 and KLF4, which induces an anti-inflammatory program that promotes vascular resilience. Here we identify clustered γ-protocadherins as therapeutically targetable, potent KLF2 and KLF4 suppressors whose upregulation contributes to ASCVD. Mechanistic studies show that γ-protocadherin cleavage results in translocation of the conserved intracellular domain to the nucleus where it physically associates with and suppresses signaling by the Notch intracellular domain. γ-Protocadherins are elevated in human ASCVD endothelium; their genetic deletion or antibody blockade protects from ASCVD in mice without detectably compromising host defense against bacterial or viral infection. These results elucidate a fundamental mechanism of vascular inflammation and reveal a method to target the endothelium rather than the immune system as a protective strategy in ASCVD.

PIEZO1 targeting in macrophages boosts phagocytic activity and foam cell apoptosis in atherosclerosis.

The rising incidences of atherosclerosis have necessitated efforts to identify novel targets for therapeutic interventions. In the present study, we observed increased expression of the mechanosensitive calcium channel Piezo1 transcript in mouse and human atherosclerotic plaques, correlating with infiltration of PIEZO1-expressing macrophages. In vitro administration of Yoda1, a specific agonist for PIEZO1, led to increased foam cell apoptosis and enhanced phagocytosis by macrophages. Mechanistically, PIEZO1 activation resulted in intracellular F-actin rearrangement, elevated mitochondrial ROS levels and induction of mitochondrial fragmentation upon PIEZO1 activation, as well as increased expression of anti-inflammatory genes. In vivo, ApoE-/- mice treated with Yoda1 exhibited regression of atherosclerosis, enhanced stability of advanced lesions, reduced plaque size and necrotic core, increased collagen content, and reduced expression levels of inflammatory markers. Our findings propose PIEZO1 as a novel and potential therapeutic target in atherosclerosis.

Lack of alpha CGRP exacerbates the development of atherosclerosis in ApoE-knockout mice.

The effects of calcitonin gene-related peptide (CGRP) on atherosclerosis remain unclear. We used apolipoprotein E-deficient (ApoE-/-) mice to generate double-knockout ApoE-/-:CGRP-/- mice lacking alpha CGRP. ApoE-/-:CGRP-/- mice exhibited larger atherosclerotic plaque areas, peritoneal macrophages with enhanced migration functions, and elevated levels of the inflammatory cytokine tumor necrosis factor (TNF)-⍺. Thus, we also explored whether inhibiting TNF-⍺ could improve atherosclerosis in ApoE-/-:CGRP-/- mice by administering etanercept intraperitoneally once a week (5 mg/kg) alongside a high-fat diet for 2 weeks. This treatment led to significant reductions in aortic root lesion size, atherosclerotic plaque area and macrophage migration in ApoE-/-:CGRP-/- mice compared with mice treated with human IgG (5 mg/kg). We further examined whether results observed in ApoE-/-:CGRP-/- mice could similarly be obtained by administering a humanized monoclonal CGRP antibody, galcanezumab, to ApoE-/- mice. ApoE-/- mice were subcutaneously administered galcanezumab at an initial dose of 50 mg/kg, followed by a dose of 30 mg/kg in the second week. Galcanezumab administration did not affect systolic blood pressure, serum lipid levels, or macrophage migration but led to a significant increase in lipid deposition at the aortic root. These findings suggest that alpha CGRP plays a critical role in inhibiting the progression of atherosclerosis.

TNF Induces Laminin-332-Encoding Genes in Endothelial Cells and Laminin-332 Promotes an Atherogenic Endothelial Phenotype.

Laminins are essential components of the basement membranes, expressed in a tissue- and cell-specific manner under physiological conditions. During inflammatory circumstances, such as atherosclerosis, alterations in laminin composition within vessels have been observed. Our study aimed to assess the influence of tumor necrosis factor-alpha (TNF), a proinflammatory cytokine abundantly found in atherosclerotic lesions, on endothelial laminin gene expression and the effects of laminin-332 (LN332) on endothelial cells' behavior. We also evaluated the expression of LN332-encoding genes in human carotid atherosclerotic plaques. Our findings demonstrate that TNF induces upregulation of LAMB3 and LAMC2, which, along with LAMA3, encode the LN332 isoform. Endothelial cells cultured on recombinant LN332 exhibit decreased claudin-5 expression and display a loosely connected phenotype, with an elevated expression of chemokines and leukocyte adhesion molecules, enhancing their attractiveness and adhesion to leukocytes in vitro. Furthermore, LAMB3 and LAMC2 are upregulated in human carotid plaques and show a positive correlation with TNF expression. In summary, TNF stimulates the expression of LN332-encoding genes in human endothelial cells and LN332 promotes an endothelial phenotype characterized by compromised junctional integrity and increased leukocyte interaction. These findings highlight the importance of basement membrane proteins for endothelial integrity and the potential role of LN332 in atherosclerosis.

Paired Transcriptomic Analyses of Atheromatous and Control Vessels Reveal Novel Autophagy and Immunoregulatory Genes in Peripheral Artery Disease.

Peripheral artery disease (PAD), a significant health burden worldwide, affects lower extremities due to atherosclerosis in peripheral vessels. Although the mechanisms of PAD have been well studied, the molecular milieu of the plaques localized within peripheral arteries are not well understood. Thus, to identify PAD-lesion-specific gene expression profiles precluding genetic, environmental, and dietary biases, we studied the transcriptomic profile of nine plaque tissues normalized to non-plaque tissues from the same donors. A total of 296 upregulated genes, 274 downregulated genes, and 186 non-coding RNAs were identified. STAG1, SPCC3, FOXQ1, and E2F3 were key downregulated genes, and CD93 was the top upregulated gene. Autophagosome assembly, cellular response to UV, cytoskeletal organization, TCR signaling, and phosphatase activity were the key dysregulated pathways identified. Telomerase regulation and autophagy were identified as novel interacting pathways using network analysis. The plaque tissue was predominantly composed of immune cells and dedifferentiated cell populations indicated by cell-specific marker-imputed gene expression analysis. This study identifies novel genes, non-coding RNAs, associated regulatory pathways, and the cell composition of the plaque tissue in PAD patients. The autophagy and immunoregulatory genes may drive novel mechanisms, resulting in atheroma. These novel interacting networks and genes have potential for PAD-specific therapeutic applications.

Aberrant mitochondrial DNA synthesis in macrophages exacerbates inflammation and atherosclerosis.

There is a large body of evidence that cellular metabolism governs inflammation, and that inflammation contributes to the progression of atherosclerosis. However, whether mitochondrial DNA synthesis affects macrophage function and atherosclerosis pathology is not fully understood. Here we show, by transcriptomic analyzes of plaque macrophages, spatial single cell transcriptomics of atherosclerotic plaques, and functional experiments, that mitochondrial DNA (mtDNA) synthesis in atherosclerotic plaque macrophages are triggered by vascular cell adhesion molecule 1 (VCAM-1) under inflammatory conditions in both humans and mice. Mechanistically, VCAM-1 activates C/EBPα, which binds to the promoters of key mitochondrial biogenesis genes - Cmpk2 and Pgc1a. Increased CMPK2 and PGC-1α expression triggers mtDNA synthesis, which activates STING-mediated inflammation. Consistently, atherosclerosis and inflammation are less severe in Apoe-/- mice lacking Vcam1 in macrophages. Downregulation of macrophage-specific VCAM-1 in vivo leads to decreased expression of LYZ1 and FCOR, involved in STING signalling. Finally, VCAM-1 expression in human carotid plaque macrophages correlates with necrotic core area, mitochondrial volume, and oxidative damage to DNA. Collectively, our study highlights the importance of macrophage VCAM-1 in inflammation and atherogenesis pathology and proposes a self-acerbating pathway involving increased mtDNA synthesis.

Identification and experimental validation of KMO as a critical immune-associated mitochondrial gene in unstable atherosclerotic plaque.

BACKGROUND: The heightened risk of cardiovascular and cerebrovascular events is associated with the increased instability of atherosclerotic plaques. However, the lack of effective diagnostic biomarkers has impeded the assessment of plaque instability currently. This study was aimed to investigate and identify hub genes associated with unstable plaques through the integration of various bioinformatics tools, providing novel insights into the detection and treatment of this condition. METHODS: Weighted Gene Co-expression Network Analysis (WGCNA) combined with two machine learning methods were used to identify hub genes strongly associated with plaque instability. The cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) method was utilized to assess immune cell infiltration patterns in atherosclerosis patients. Additionally, Gene Set Variation Analysis (GSVA) was conducted to investigate the potential biological functions, pathways, and mechanisms of hub genes associated with unstable plaques. To further validate the diagnostic efficiency and expression of the hub genes, immunohistochemistry (IHC), quantitative real-time polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) were performed on collected human carotid plaque and blood samples. Immunofluorescence co-staining was also utilized to confirm the association between hub genes and immune cells, as well as their colocalization with mitochondria. RESULTS: The CIBERSORT analysis demonstrated a significant decrease in the infiltration of CD8 T cells and an obvious increase in the infiltration of M0 macrophages in patients with atherosclerosis. Subsequently, two highly relevant modules (blue and green) strongly associated with atherosclerotic plaque instability were identified. Through intersection with mitochondria-related genes, 50 crucial genes were identified. Further analysis employing least absolute shrinkage and selection operator (LASSO) logistic regression and support vector machine recursive feature elimination (SVM-RFE) algorithms revealed six hub genes significantly associated with plaque instability. Among them, NT5DC3, ACADL, SLC25A4, ALDH1B1, and MAOB exhibited positive correlations with CD8 T cells and negative correlations with M0 macrophages, while kynurenine 3-monooxygenas (KMO) demonstrated a positive correlation with M0 macrophages and a negative correlation with CD8 T cells. IHC and RT-qPCR analyses of human carotid plaque samples, as well as ELISA analyses of blood samples, revealed significant upregulation of KMO and MAOB expression, along with decreased ALDH1B1 expression, in both stable and unstable samples compared to the control samples. However, among the three key genes mentioned above, only KMO showed a significant increase in expression in unstable plaque samples compared to stable plaque samples. Furthermore, the expression patterns of KMO in human carotid unstable plaque tissues and cultured mouse macrophage cell lines were assessed using immunofluorescence co-staining techniques. Finally, lentivirus-mediated KMO silencing was successfully transduced into the aortas of high-fat-fed ApoE-/- mice, with results indicating that KMO silencing attenuated plaque formation and promoted plaque stability in ApoE-/- mice. CONCLUSIONS: The results suggest that KMO, a mitochondria-targeted gene associated with macrophage cells, holds promise as a valuable diagnostic biomarker for assessing the instability of atherosclerotic plaques.

From metabolic to epigenetic: Insight into trained macrophages in atherosclerosis (Review).

Atherosclerosis (AS) is a chronic inflammatory disease caused by the deposition of lipoproteins and sequent immune responses. Within the atherosclerotic plaque, macrophages are the most abundant immune cells and play a great part as protagonists and promoters of AS. In the past decade, the concept of 'trained immunity' has emerged, which highlights the memory characteristics of innate immunity, thus opening up a new avenue of research. Evidence suggests that trained immunity may regulate the onset and progression of AS with trained macrophages playing an important and dynamic role in atherogenesis. The present review provided a summary of concepts related to trained immunity and its relationship with AS. Furthermore, different phenotypes of macrophages responding to various stimuli within the atherosclerotic plaque were presented, along with the complex mechanisms of metabolic and epigenetic reprogramming in the cells. Finally, several promising therapeutic approaches for AS cardiovascular disease were discussed, which may shed light on new clinical strategies.

Associations of genetically predicted vitamin D status and deficiency with the risk of carotid artery plaque: a Mendelian randomization study.

Low concentrations of circulating 25-hydroxy-vitamin D are observationally associated with an increased risk of subclinical atherosclerosis and cardiovascular disease. However, randomized controlled trials have not reported the beneficial effects of vitamin D supplementation on atherosclerotic cardiovascular disease (ASCVD) outcomes. Whether genetically predicted vitamin D status confers protection against the development of carotid artery plaque, a powerful predictor of subclinical atherosclerosis, remains unknown. We conducted a two-sample Mendelian randomization (MR) study to explore the association of genetically predicted vitamin D status and deficiency with the risk of developing carotid artery plaque. We leveraged three genome-wide association studies (GWAS) of vitamin D status and one GWAS of vitamin D deficiency. We used the inverse-variance weighted (IVW) approach as our main method, and MR-Egger, weighted-median, and radialMR as MR sensitivity analyses. We also conducted sensitivity analyses using biologically plausible genetic instruments located within genes encoding for vitamin D metabolism (GC, CYP2R1, DHCR7, CYP24A1). We did not find significant associations between genetically predicted vitamin D status (Odds ratio (OR) = 0.99, P = 0.91) and deficiency (OR = 1.00, P = 0.97) with the risk of carotid artery plaque. We additionally explored the potential causal effect of vitamin D status on coronary artery calcification (CAC) and carotid intima-media thickness (cIMT), two additional markers of subclinical atherosclerosis, and we did not find any significant association (ßCAC = - 0.14, P = 0.23; ßcIMT = 0.005, P = 0.19). These findings did not support the causal effects of vitamin D status and deficiency on the risk of developing subclinical atherosclerosis.

Smooth muscle NF90 deficiency ameliorates diabetic atherosclerotic calcification in male mice via FBXW7-AGER1-AGEs axis.

Hyperglycemia accelerates calcification of atherosclerotic plaques in diabetic patients, and the accumulation of advanced glycation end products (AGEs) is closely related to the atherosclerotic calcification. Here, we show that hyperglycemia-mediated AGEs markedly increase vascular smooth muscle cells (VSMCs) NF90/110 activation in male diabetic patients with atherosclerotic calcified samples. VSMC-specific NF90/110 knockout in male mice decreases obviously AGEs-induced atherosclerotic calcification, along with the inhibitions of VSMC phenotypic changes to osteoblast-like cells, apoptosis, and matrix vesicle release. Mechanistically, AGEs increase the activity of NF90, which then enhances ubiquitination and degradation of AGE receptor 1 (AGER1) by stabilizing the mRNA of E3 ubiquitin ligase FBXW7, thus causing the accumulation of more AGEs and atherosclerotic calcification. Collectively, our study demonstrates the effects of VSMC NF90 in mediating the metabolic imbalance of AGEs to accelerate diabetic atherosclerotic calcification. Therefore, inhibition of VSMC NF90 may be a potential therapeutic target for diabetic atherosclerotic calcification.