Human Atherosclerotic Plaques Research Articles

Abstract 4132752: The immune checkpoint landscape in human atherosclerosis: influence of lipid lowering and type 2 diabetes

Immune checkpoint inhibitor (ICI) therapies substantially increase the risk of atherosclerotic cardiovascular events in cancer survivors. Here, we mapped the expression of immune checkpoints (ICs) within human atherosclerotic plaques, unraveling an intricate local network of immune cell-cell interactions in the context of relevant comorbidities like dyslipidemia and diabetes. Our findings identified a population of mature, regulatory CCR7 + FSCN1 + dendritic cells as pivotal orchestrators of IC-mediated signaling within human and murine plaques, a function akin to their role in tumor environments. In clinical cohorts, type 2 diabetes diminished CCR7 + FSCN1 + dendritic cells within atherosclerotic plaques and dampened interactions steered by PD-1 or CTLA4 consistently across immune cell subpopulations. This effect was also present and further decreased by lipid lowering in the circulation, where CD86–CTLA4 was among the most prominent IC pairs signaling between regulatory T cells and myeloid populations including CCR7 + FSCN1 + dendritic cells. These findings underline the vulnerability of atherosclerotic plaques to cancer immunotherapies and underscore the critical impact of cardiometabolic disease and lipid-lowering strategies on the atherosclerotic IC landscape, offering a framework toward the reshaping of clinical approaches.

Abstract 4145859: Clonal Hematopoiesis of Indeterminate Potential Increases Mortality Risk in Coronary Artery Disease

Background: Clonal hematopoiesis of indeterminate potential (CHIP) has been associated with an increased incidence of common late-onset diseases including myocardial infarction. Research Question: Here we examined the impact of CHIP on mortality in patients with coronary artery disease (CAD) and further explored potential underlying mechanisms. Methods: CAD patients were examined for CHIP-defining mutations using deep DNA sequencing. Propensity score matching was used to compare CHIP CAD with non-CHIP CAD patients. Survival was analyzed using Kaplan-Meier curves and Cox proportional hazard models. Functional aspects were investigated at the protein and RNA level in cardiovascular relevant human postmortem tissue from the Munich cardIovaScular StudIes biObaNk (MISSION), in CAD patients from an independent cohort and in in-vitro macrophage models. Results: Within 7,933 CAD patients screened by deep-DNA-sequencing with a 13 gene panel, 2,002 carried at least one CHIP defining mutation with a variant allele frequency ≥2%. After 1:1 matching with non-CHIP carriers we revealed a significantly elevated 3-year mortality in the group of CHIP carriers [HR 1.48 (CI 95% 1.23-1.78); p<0.0001)] as well as in carriers of mutations in one out of seven individual genes – ASXL1 , DNMT3A , PPM1D , SF3B1 , SRSF2 , TET2 , and U2AF1 . In human coronary atherosclerotic plaque we visualized CHIP-mutations within leukocytes. Further, within coronary artery plaque of deceased TET2 mutation carriers (n=26 vs. n=13 controls) we identified by proteomic profiling an upregulation of inflammatory and metabolic pathways, including enhanced lipid metabolism. In an independent cohort of CAD patients undergoing coronary bypass surgery, RNA sequencing data from monocyte-derived macrophages revealed in TET2 CHIP carriers associations with CAD complexity. TET2 -mutated macrophages studied in-vitro revealed an increased uptake of low-density lipoprotein (LDL) and oxidized LDL-cholesterol besides a known upregulation of inflammatory pathways. Conclusion: In summary, this study emphasizes CHIP as a significant prognostic factor in CAD patients, associated with increased complexity of CAD and altered gene expression within human coronary artery plaques. Mechanistically, TET2 mutations modify the behavior of CHIP-affected macrophages, leading to heightened inflammation and increased uptake of LDL-cholesterol.

Abstract 4137846: Smooth muscle expression of RNA editing enzyme ADAR1 controls vascular integrity and progression of atherosclerosis

Mapping the genomic architecture of complex disease has been predicated on the understanding that genetic variants influence disease risk through modifying gene expression. However, recent discoveries have revealed that a significant burden of disease heritability in common autoinflammatory disorders and coronary artery disease is mediated through genetic variation modifying post-transcriptional modification of RNA through adenosine-to-inosine (A-to-I) RNA editing. This common RNA modification is catalyzed by ADAR enzymes, where ADAR1 edits specific immunogenic double stranded RNA (dsRNA) to prevent activation of the double strand RNA (dsRNA) sensor MDA5 ( IFIH1 ) and stimulation of an interferon stimulated gene (ISG) response. Multiple lines of human genetic data indicate impaired RNA editing and increased dsRNA sensing to be an important mechanism of coronary artery disease (CAD) risk. Here, we provide a crucial link between observations in human genetics and mechanistic cell biology leading to progression of CAD. Through analysis of human atherosclerotic plaque, we implicate the vascular smooth muscle cell (SMC) to have a unique requirement for RNA editing, and that ISG induction occurs in SMC phenotypic modulation, implicating MDA5 activation. Through culture of human coronary artery SMCs, generation of a conditional SMC specific Adar1 deletion mouse model on a pro-atherosclerosis background, and with incorporation of single cell RNA sequencing cellular profiling, we further show that Adar1 controls SMC phenotypic state, is required to maintain vascular integrity, and controls progression of atherosclerosis and vascular calcification. Our data implicates MDA5 activation to occur in SMC phenotypic modulation and accelerates formation of chondromyocytes in a distinct cellular lineage trajectory — indicating a cell type and context specific requirement of RNA editing. Through this work, we describe a fundamental new mechanism of CAD, where RNA editing and sensing of dsRNA in a cell type and context specific mechanism mediates disease progression, bridging our understanding of human genetics and disease causality.

The alpha 7 nicotinic acetylcholine receptor agonist PHA 568487 dampens inflammation in PBMCs from patients with newly discovered coronary artery disease.

The alpha 7 nicotinic acetylcholine receptor (α7nAChR) regulates inflammation in experimental models and is expressed in human peripheral blood mononuclear cells (PBMCs) and in human atherosclerotic plaques. However, its role in regulating inflammation in patients with cardiovascular disease is unknown. This study aims to investigate whether α7nAChR stimulation can reduce the inflammatory response in PBMCs from patients with newly diagnosed coronary artery disease (CAD). Human PBMCs, extracted from patients with verified CAD (n = 38) and control participants with healthy vessels (n = 38), were challenged in vitro with lipopolysaccharide (LPS) in combination with the α7nAChR agonist PHA 568487. Cytokine levels of the supernatants were analyzed using a multiplex immunoassay. Patients in the CAD group were reexamined after 6 mo. The immune response to LPS did not differ between PBMCs from control and CAD groups. α7nAChR stimulation decreased TNFα in both control and CAD groups. The most pronounced effect of α7nAChR stimulation was observed in patients with CAD at their first visit, where 15 of 17 cytokines were decreased [IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 (p70), IL-17A, G-CSF, GM-CSF, IFN-γ, MCP-1, MIP-1β, and TNFα]. In conclusion, stimulation with α7nAChR agonist PHA 568487 dampens the inflammatory response in human PBMCs. This finding suggests that the anti-inflammatory properties of the α7nAChR may have a role in treating CAD.NEW & NOTEWORTHY The α7nAChR is an important regulator of inflammation; however, its anti-inflammatory function in patients with newly diagnosed coronary artery disease (CAD) remains unclear. We demonstrate that stimulation of α7nAChR with PHA 568487 attenuates the inflammatory response in immune cells extracted from healthy controls and patients with newly diagnosed CAD, with a more pronounced effect observed in patients with CAD. This suggests that the anti-inflammatory properties of α7nAChR may have a role in treating chronic inflammatory diseases.

Plasma levels of sCASP3 predicts carotid intima media thickness progression

Abstract Background Studies on human atherosclerotic plaques have shown that apoptosis is associated with rupture prone plaques and that apoptosis is a key process for fast plaque progression. In line with this, we showed that circulating levels of soluble Caspase-3 (sCaspase-3) predict atherosclerotic events. However, the relationship between circulating levels of apoptosis markers and plaque progression remains to be explored. Purpose Here we aimed to investigate if circulating levels of apoptosis markers could predict mean intima-media thickness in the common carotid artery (IMT-CCA) progression among individuals with atherosclerotic carotid plaques. Methods The Malmö Diet and Cancer (MDC) cardiovascular cohort was used. MDC is a prospective population-based cohort study. Baseline variables (blood, clinical information and carotid ultrasound measurements) were collected between 1991-1994 and the right carotid ultrasound re-examinations were performed between 1994-2001. IMT was measured at by ultrasound at baseline as well as at the re-examinations. The Olink Proximity Extension Assay (SciLifeLab, Uppsala, Sweden) was used to analyze plasma levels of sCaspase-3, sFADD, sCaspase-8, sTRAIL-R2 and sTNFR1 at baseline. Correlation and multiple linear regression analyses, adjusted for age, sex, and cardiovascular risk factors, were employed to evaluate the relationships between apoptosis biomarkers and IMT-CCA as well as average annual change in IMT-CCA. Results In total, 1929 participants (median age 60 years) with an ultrasound confirmed carotid plaque (focal IMT >1.2mm) at baseline were included. Subjects who underwent ultrasound IMT re-examination over a period of >4.5 years were divided into a discovery (n=406) and a validation (n=355) cohort. sCaspase-3 was identified as the marker with the strongest correlation to IMT-CCA at baseline (p=0.02) and upon re-examination (p=0.04), among the five apoptosis markers examined, in both the discovery and validation cohorts. sCaspase-3 also showed a correlation with the average annual change of IMT-CCA (r=0.12, p=8×10-4) in both cohorts. Furthermore, when diving the cohort into tertiles based on plasma levels of sCapase-3, subjects with high levels of sCaspase-3 (3rd tertile) had greater annual changes in IMT-CCA compared to the 1st tertile (p=0.03). Multiple linear regression analyses confirmed that sCapase-3 was associated with the average annual IMT-CCA change in both the discovery (beta=0.14, 95% confidence interval (CI) 0.04–0.24) cohort and the validation cohort (beta=0.11, 95%CI 0.01–0.21). Conclusion Using a large, population-based study with repeated IMT measurements, we demonstrated that circulating levels of sCapase-3 were associated with increasing IMT in the CCA. While further studies are warranted, these results suggest that sCapase-3 levels could serve as potential screening marker to identify individuals at higher risk for plaque progression.

Immunohistochemical Localization of Fibroblast Activation Protein in Coronary Arteries with Different Forms of Atherosclerosis

Background: Fibroblast activation protein (FAP) is a cell surface glycoprotein expressed by myofibroblasts in areas of active tissue remodeling. It plays a potentially important role in cardiac remodeling, atherosclerotic plaque formation, and plaque rupture. Given the distinct pathophysiology and morphology of different forms of atherosclerosis, we analyzed FAP expression in human coronary vessels with no coronary artery disease, atherosclerotic plaques at different levels of progression, and other distinct forms of coronary disease in post bypass vein grafting and cardiac allograft vasculopathy after a heart transplant. Methods: Immunohistochemical staining with monoclonal F19 mouse anti-human FAP antibody was performed to identify FAP in human atherosclerotic plaques, coronary bypass atherosclerosis, and post-transplant arteriosclerosis. The presence and distribution of FAP in different types and stages of human atherosclerosis were compared. Results: There was no FAP staining in patients with no significant coronary disease. All different types of human atherosclerotic lesioning lesions showed the presence of FAP expression, with different staining patterns in advanced atherosclerotic plaque, vein graft atherosclerosis lesions, and arteriosclerosis after a heart transplant. Conclusions: These data suggest that FAP may be a potential diagnostic marker and target for interventions, not only in coronary atherosclerotic plaque, but also in other forms of coronary disease, which have distinct pathophysiologies and currently limited treatment options.

Olfr2-positive macrophages originate from monocytes proliferate in situ and present a pro-inflammatory foamy-like phenotype.

Olfactory receptor 2 (Olfr2) has been identified in a minimum of 30% of vascular macrophages, and its depletion was shown to reduce atherosclerosis progression. Mononuclear phagocytes, including monocytes and macrophages within the vessel wall, are major players in atherosclerosis. Single-cell RNA sequencing studies revealed that atherosclerotic artery walls encompass several monocytes and vascular macrophages, defining at least nine distinct subsets potentially serving diverse functions in disease progression. This study investigates the functional phenotype and ontogeny of Olfr2-expressing vascular macrophages in atherosclerosis. Olfr2+ macrophages rapidly increase in Apoe-/- mice's aorta when fed a Western diet (WD). Mass cytometry showed that Olfr2+ cells are clustered within the CD64 high population and enriched for CD11c and Ccr2 markers. Olfr2+ macrophages express many pro-inflammatory cytokines, including Il1b, Il6, Il12, and Il23, and chemokines, including Ccl5, Cx3cl1, Cxcl9, and Ccl22. By extracting differentially expressed genes from bulk RNA sequencing (RNA-seq) of Olfr2+ vs. Olfr2- macrophages, we defined a signature that significantly mapped to single-cell data of plaque myeloid cells, including monocytes, subendothelial MacAir, and Trem2Gpnmb foamy macrophages. By adoptive transfer experiments, we identified that Olfr2 competent monocytes from CD45.1Apoe-/-Olfr2+/+ mice transferred into CD45.2Apoe-/-Olfr2-/- recipient mice fed WD for 12 weeks, accumulate in the atherosclerotic aorta wall already at 72 h, and differentiate in macrophages. Olfr2+ macrophages showed significantly increased BrdU incorporation compared to Olfr2- macrophages. Flow cytometry confirmed that at least 50% of aortic Olfr2+ macrophages are positive for BODIPY staining and have increased expression of both tumour necrosis factor and interleukin 6 compared to Olfr2- macrophages. Gene set enrichment analysis of the Olfr2+ macrophage signature revealed a similar enrichment pattern in human atherosclerotic plaques, particularly within foamy/TREM2hi-Mφ and monocytes. In summary, we conclude that Olfr2+ macrophages in the aorta originate from monocytes and can accumulate at the early stages of disease progression. These cells can undergo differentiation into MacAir and Trem2Gpnmb foamy macrophages, exhibiting proliferative and pro-inflammatory potentials. This dynamic behaviour positions them as key influencers in shaping the myeloid landscape within the atherosclerotic plaque.

Abstract P454: Screening of risk genes for carotid vulnerable plaque based on transcriptomics and experimental verification

Purpose and background: This study aims to conduct an in-depth analysis of human carotid atherosclerotic plaques using transcriptomic data to elucidate the relationship between hypoxia-related genes and the stability of carotid artery plaques and explore their connection with immune infiltration. Methods: Four GEO datasets (GSE21545, GSE24495, GSE159677, and GSE189300) were used. Initially, gene expression data from carotid artery plaque samples in GSE21545 and GSE24495 were analyzed. Hypoxia-related gene sets were obtained from MSigDB, and the "HALLMARK_HYPOXIA" gene set was explicitly selected. GSEA software was used for ultimately selecting CDKN1C and HAS1 as characteristic genes. Subsequently, sample clustering analysis was conducted to determin the optimal number of clusters. Gene set enrichment and differential analysis were performed. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting experiments were conducted for preliminary validation of CDKN1C and HAS1 expression levels in ApoE knockout mice at 16 and 6 weeks. Subsequently, single-cell data from GSE159677 were processed to determine the differential localization of CDKN1C in carotid atherosclerotic plaques, with functional analysis conducted using GSE189300. Results: CDKN1C and HAS1 genes were found to exhibit differential expression in plaques and were correlated with hypoxia and immune scores. Clustering analysis revealed two main clusters, with patients in cluster A showing a higher risk of adverse events and a stronger association with immunity. Genes with a positive effect on atherosclerosis were highly expressed in poorer prognosis cluster A. Validation dataset GSE24495 also confirmed the grouping ability of CDKN1C and HAS1 genes, showing enrichment in atherosclerosis-related pathways. RT-qPCR and protein immunoblotting experiments demonstrated no significant differences in HAS1 expression levels, while CDKN1C showed lower protein expression in 16-week ApoE knockout mice compared to 6 weeks. Single-cell data from GSE159677 localized differential expression of CDKN1C in plaque cells. Finally, immunofluorescence validation confirmed the coexistence of CDKN1C and vascular smooth muscle cells. Conclusion: This study comprehensively analyzed transcriptomics data from carotid atherosclerotic plaques, revealing the significant roles of CDKN1C and HAS1 genes. Immunofluorescence validation further supported the crucial role of these genes in carotid atherosclerosis.

H19 lncRNA overexpression in human atherosclerotic plaques and plasma: Potential biomarker from RNA-Seq analysis

H19 lncRNA overexpression in human atherosclerotic plaques and plasma: Potential biomarker from RNA-Seq analysis

The landscape of somatic mutations in human atherosclerotic plaques indicates the presence of large clonal cell populations

The landscape of somatic mutations in human atherosclerotic plaques indicates the presence of large clonal cell populations

MIRO1 controls energy production and proliferation of smooth muscle cells.

The outer mitochondrial Rho GTPase 1, MIRO1, mediates mitochondrial motility within cells, but implications for vascular smooth muscle cell (VSMC) physiology and its roles invascular diseases, such as neointima formation following vascular injury are widely unknown. An in vivo model of selective Miro1 deletion in VSMCs was generated, and the animals were subjected to carotid artery ligation. The molecular mechanisms relevant to VSMC proliferation were then explored in explanted VSMCs by imaging mitochondrial positioning and cristae structure and assessing the effects on ATP production, metabolic function and interactions with components of the electron transport chain (ETC). MIRO1 was robustly expressed in VSMCs within human atherosclerotic plaques and promoted VSMC proliferation and neointima formation in mice by blocking cell-cycle progression at G1/S, mitochondrial positioning, and PDGF-induced ATP production and respiration; overexpression of a MIRO1 mutant lacking the EF hands that are required for mitochondrial mobility did not fully rescue these effects. At the ultrastructural level, Miro1 deletion distorted the mitochondrial cristae and reduced the formation of super complexes and the activity of ETC complex I. Mitochondrial motility is essential for VSMC proliferation and relies on MIRO1. The EF-hands of MIRO1 regulate the intracellular positioning of mitochondria. Additionally, the absence of MIRO1 leads to distorted mitochondrial cristae and reduced ATP generation. Our findings demonstrate that motility is linked to mitochondrial ATP production. We elucidated two unrecognized mechanisms through which MIRO1 influences cell proliferation by modulating mitochondria: first, by managing mitochondrial placement via Ca2+-dependent EF hands, and second, by affecting cristae structure and ATP synthesis.

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.

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.

Hypoxia activates macrophage-NLRP3 inflammasome promoting atherosclerosis via PFKFB3-driven glycolysis.

The onset and progression of atherosclerosis are closely linked to the involvement of macrophages. While the contribution of NLRP3 inflammasome activation to the creation of a local highly inflammatory microenvironment is well recognized, the precise triggers remain unclear. In this study, we aimed to investigate the regulatory mechanism of NLRP3 inflammasome activation in response to hypoxia-induced glycolysis involving PFKFB3 in the development of atherosclerosis. To develop an atherosclerosis model, we selected ApoE knockout mice treated with a high-fat western diet. We then quantified the expression of HIF-1α, PFKFB3, and NLRP3. In addition, we administered the PFKFB3 inhibitor PFK158 during atherosclerosis modeling. The glycolytic activity was subsequently determined through 18F-FDG micro-PET/CT, exvivo glucose uptake, and ECAR analysis. Furthermore, we employed lipopolysaccharide (LPS) and TNF-α to induce the differentiation of bone marrow-derived macrophages (BMDMs) into M1-like phenotypes under both hypoxic and normoxic conditions. Our histological analyses revealed the accumulation of PFKFB3 in human atherosclerotic plaques, demonstrating colocalization with NLRP3 expression and macrophages. Treatment with PFK158 reduced glycolytic activity and NLRP3 inflammasome activation, thereby mitigating the occurrence of atherosclerosis. Mechanistically, hypoxia promoted glycolytic reprogramming and NLRP3 inflammasome activation in BMDMs. Subsequent blocking of either HIF-1α or PFKFB3 downregulated the NLRP3/Caspase-1/IL-1β pathway in hypoxic BMDMs. Our study demonstrated that the HIF-1α/PFKFB3/NLRP3 axis serves as a crucial mechanism for macrophage inflammation activation in the emergence of atherosclerosis. The therapeutic potential of PFKFB3 inhibition may represent a promising strategy for atheroprotection.

Multiomics unveils extracellular vesicle-driven mechanisms of endothelial communication in human carotid atherosclerosis.

Background: Carotid atherosclerosis is orchestrated by cell-cell communication that drives progression along a clinical continuum (asymptomatic to symptomatic). Extracellular vesicles (EVs) are cell-derived nanoparticles representing a new paradigm in cellular communication. Little is known about their biological cargo, cellular origin/destination, and functional roles in human atherosclerotic plaque. Methods: EVs were enriched via size exclusion chromatography from human carotid endarterectomy samples dissected into paired plaque and marginal zones (symptomatic n=16, asymptomatic n=13). EV cargos were assessed via whole transcriptome miRNA sequencing and mass spectrometry-based proteomics. EV multi-omics were integrated with bulk and single cell RNA-sequencing (scRNA-seq) datasets to predict EV cellular origin and ligand-receptor interactions, and multi-modal biological network integration of EV-cargo was completed. EV functional impact was assessed with endothelial angiogenesis assays. Results: Carotid plaques contained more EVs than adjacent marginal zones, with differential enrichment for EV-miRNAs and EV-proteins in key atherogenic pathways. EV cellular origin analysis suggested that tissue EV signatures originated from endothelial cells (EC), smooth muscle cells (SMC), and immune cells. Integrated tissue vesiculomics and scRNA-seq indicated complex EV-vascular cell communication that changed with disease progression and plaque vulnerability (i.e., symptomatic disease). Plaques from symptomatic patients, but not asymptomatic patients, were characterized by increased involvement of endothelial pathways and more complex ligand-receptor interactions, relative to their marginal zones. Plaque-EVs were predicted to mediate communication with ECs. Pathway enrichment analysis delineated an endothelial signature with roles in angiogenesis and neovascularization - well-known indices of plaque instability. This was validated functionally, wherein human carotid symptomatic plaque EVs induced sprouting angiogenesis in comparison to their matched marginal zones. Conclusion: Our findings indicate that EVs may drive dynamic changes in plaques through EV- vascular cell communication and effector functions that typify vulnerability to rupture, precipitating symptomatic disease. The discovery of endothelial-directed angiogenic processes mediated by EVs creates new therapeutic avenues for atherosclerosis.

Cross-species single-cell RNA sequencing reveals divergent phenotypes and activation states of adaptive immunity in human carotid and experimental murine atherosclerosis.

The distinct functions of immune cells in atherosclerosis have been mostly defined by preclinical mouse studies. Contrastingly, the immune cell composition of human atherosclerotic plaques and their contribution to disease progression is only poorly understood. It remains uncertain whether genetic animal models allow for valuable translational approaches. Single cell RNA-sequencing (scRNA-seq) was performed to define the immune cell landscape in human carotid atherosclerotic plaques. The human immune cell repertoire demonstrated an unexpectedly high heterogeneity and was dominated by cells of the T-cell lineage, a finding confirmed by immunohistochemistry. Bioinformatical integration with 7 mouse scRNA-seq data sets from adventitial and atherosclerotic vascular tissue revealed a total of 51 identities of cell types and differentiation states, of which some were only poorly conserved between species and exclusively found in humans. Locations, frequencies, and transcriptional programs of immune cells in mouse models did not resemble the immune cell landscape in human carotid atherosclerosis. In contrast to standard mouse models of atherosclerosis, human plaque leukocytes were dominated by several T-cell phenotypes with transcriptional hallmarks of T-cell activation and memory formation, T-cell receptor-, and pro-inflammatory signaling. Only mice at the age of 22 months partially resembled the activated T-cell phenotype. In a validation cohort of 43 patients undergoing carotid endarterectomy, the abundance of activated immune cell subsets in the plaque defined by multi-color flow cytometry associated with the extend of clinical atherosclerosis. Integrative scRNA-seq reveals a substantial difference in the immune cell composition of murine and human carotid atherosclerosis - a finding that questions the translational value of standard mouse models for adaptive immune cell studies. Clinical associations suggest a specific role for T-cell driven (auto-) immunity in human plaque formation and -instability.

Clustering Methods for the Characterization of Synchrotron Radiation X‐Ray Fluorescence Images of Human Carotid Atherosclerotic Plaque

This study employs computational algorithms to automatically identify and classify features in X‐Ray fluorescence (XRF) microscopy images. Principal component analysis (PCA) and unsupervised machine learning algorithms, such as Gaussian mixture (GM) clustering, are implemented to label features on a collection of XRF maps of human atherosclerotic plaque samples. The investigation involves the hard X‐Ray nanoprobe (NanoMAX) at MAX IV synchrotron radiation facility, utilizing scanning transmission X‐Ray microscopy (STXM) and XRF techniques. The analysis covers regions of interest scanned by the beam with a step size of 200 nm, yielding XRF maps of elements like calcium, iron, and zinc. These maps reveal intricate structures unsuitable for manual labeling. However, they can be accurately classified in an automated fashion using GM. Prior to clustering, PCA is used to deal with repeated patterns and background areas. The resulting clusters are associated with different types of features, which can be identified as specific tissues confirmed by histology. Regions of high concentrations of phosphorus, sulfur, calcium, and iron are found in the samples. These regions are also observed in the STXM results as spots of low transmission that typically are associated with calcium deposits only.

Flow shear force destabilizes carotid plaques by affecting CHOP and GRP78 proteins

BackgroundVarious factors, including blood, inflammatory, infectious, and immune factors, can cause ischemic stroke. However, the primary cause is often the instability of cervical arteriosclerosis plaque. It is estimated that 18-25% of ischemic strokes are caused by the rupture of carotid plaque.1 Plaque stability is crucial in determining patient prognosis. Developing a highly accurate, non-invasive, or minimally invasive technique to assess carotid plaque stability is crucial for diagnosing and treating stroke.Previous research by our group has demonstrated that the expression levels of CHOP (C/EBP homologous protein) and GRP78 (glucose-regulated protein 78) are correlated with the stability of atherosclerotic plaques.2 ObjectThis research assesses changes in GRP78 and CHOP expressions in human umbilical vein endothelial cells(HUVEC) following experiments within the hemodynamic influencing factors test system. Additionally, it includes conducting an empirical study on the impact of blood flow shear force on the stability of human carotid atherosclerotic plaques. The objective is to explore the implications of blood flow shear force on the stability of carotid atherosclerotic plaques. MethodThe hemodynamic influencing factors test bench system was configured with low (Group A, 4 dyns/cm²), medium (Group B, 8 dyns/cm²), and high shear force groups (Group C, 12 dyns/cm²). Relative expression levels of GRP78 and CHOP proteins in human umbilical vein endothelial cells were measured using Western blot analysis, and quantitative analysis of GRP78 and CHOP mRNA was conducted using RT-qPCR. Meanwhile, plaques from 60 carotid artery patients, retrieved via Carotid Endarterectomy (CEA), were classified into stable (S) and unstable (U) groups based on pathological criteria. Shear force at the carotid bifurcation was measured preoperatively using ultrasound. Western blot and RT-qPCR were used to analyze the relative expression levels of GRP78 and CHOP proteins and mRNA, respectively, in the plaque specimens from both groups. ResultExpression levels of GRP78, CHOP proteins, and their mRNAs were assessed in groups A, B, and C via Western blot and RT-qPCR. Results showed that in the low-shear group, all markers were elevated in group A compared to groups B and C. Statistical analysis revealed significantly lower shear forces at the carotid bifurcation in group U compared to group S. In group U plaques, GRP78 and CHOP expressions were significantly higher in group U than in group S. ConclusionBlood flow shear forces variably affect the expression of GRP78 and CHOP proteins, as well as their mRNA levels, in vascular endothelial cells. The lower the shear force and fluid flow rate, the higher the expression of GRP78 and CHOP, potentially leading to endoplasmic reticulum stress(ERS), which may destabilize the plaque.

Genome-scale metabolic network of human carotid plaque reveals the pivotal role of glutamine/glutamate metabolism in macrophage modulating plaque inflammation and vulnerability

BackgroundMetabolism is increasingly recognized as a key regulator of the function and phenotype of the primary cellular constituents of the atherosclerotic vascular wall, including endothelial cells, smooth muscle cells, and inflammatory cells. However, a comprehensive analysis of metabolic changes associated with the transition of plaque from a stable to a hemorrhaged phenotype is lacking.MethodsIn this study, we integrated two large mRNA expression and protein abundance datasets (BIKE, n = 126; MaasHPS, n = 43) from human atherosclerotic carotid artery plaque to reconstruct a genome-scale metabolic network (GEM). Next, the GEM findings were linked to metabolomics data from MaasHPS, providing a comprehensive overview of metabolic changes in human plaque.ResultsOur study identified significant changes in lipid, cholesterol, and inositol metabolism, along with altered lysosomal lytic activity and increased inflammatory activity, in unstable plaques with intraplaque hemorrhage (IPH+) compared to non-hemorrhaged (IPH−) plaques. Moreover, topological analysis of this network model revealed that the conversion of glutamine to glutamate and their flux between the cytoplasm and mitochondria were notably compromised in hemorrhaged plaques, with a significant reduction in overall glutamate levels in IPH+ plaques. Additionally, reduced glutamate availability was associated with an increased presence of macrophages and a pro-inflammatory phenotype in IPH+ plaques, suggesting an inflammation-prone microenvironment.ConclusionsThis study is the first to establish a robust and comprehensive GEM for atherosclerotic plaque, providing a valuable resource for understanding plaque metabolism. The utility of this GEM was illustrated by its ability to reliably predict dysregulation in the cholesterol hydroxylation, inositol metabolism, and the glutamine/glutamate pathway in rupture-prone hemorrhaged plaques, a finding that may pave the way to new diagnostic or therapeutic measures.

Zinc finger translocation‑associated protein promotes ferroptosis through the upregulation of ACSL4 expression in vascular endothelial cells.

Numerous studies have reported the potential involvement of ferroptosis in the development of atherosclerosis (AS). Acyl-CoA synthetase long chain family member 4 (ACSL4) is an essential component in the promotion of ferroptosis. The present study aimed to investigate the role of ACSL4 and zinc finger translocation-associated protein (ZFTA) in the regulation of endothelial cell ferroptosis in AS. Human umbilical vein endothelial cells (HUVECs) with ACSL4 knockout were generated using CRISPR/Cas9 technology. To assess ferroptosis, malondialdehyde concentration, iron content and reactive oxygen species levels were quantified in the present study. In addition, western blot analysis was conducted to explore the potential mechanisms underlying ACSL4 and ZFTA in the modulation of ferroptosis in HUVECs. The results of the present study demonstrated that the expression levels of ACSL4 and ZFTA were significantly increased in human atherosclerotic plaques. In addition, ACSL4 knockout led to a reduced susceptibility to ferroptosis, while ZFTA contributed to ferroptosis in HUVECs. Results of the present study also demonstrated that ZFTA overexpression upregulated ACSL4 expression in HUVECs, whereas ZFTA knockdown led to decreased ACSL4 expression. Co-transfection experiments demonstrated that the ZTFA overexpression-mediated increase in ferroptosis was reversed following ACSL4 knockdown. Collectively, results of the present study highlighted that ACSL4 mediated the effects of ZFTA on the ferroptosis of HUVECs. Thus, the present study demonstrated the potential role of ACSL4 and ZFTA in the regulation of ferroptosis, and highlighted that ferroptosis-related pathways may act as potential targets in the treatment of AS.