Macrophages In Atherosclerotic Lesions Research Articles

DNMT3A CHIP-driver mutations ignite the bone marrow

Abstract Background Clonal hematopoiesis of indeterminate potential (CHIP) is an acquired, genetic cardiovascular (CV) risk factor affecting about 10% of the general population at the age of 70 years. Most CHIP-driver mutations affect the epigenetic regulator gene DNMT3A. Experimental studies suggest a causal link between CHIP and inflammation-driven atherosclerosis, but the underlying mechanisms of how few mutant clones provoke adverse clinical outcomes in patients remain obscure. Methods Single monocytes and bone marrow cells from DNMT3A-mutation carriers undergoing coronary angiography and open-heart surgery underwent RNA and genomic DNA sequencing in parallel on a single cell level. This approach enabled us to compare mutant and non-mutant cells directly within carriers, and to non-carrier cells. Atherosclerotic chimeric mice reconstituted with a mixture of Dnmt3a-deficient (CD45.2) and -competent (CD45.1) bone marrow served as an experimental model to compare mutant to non-mutant macrophages in atherosclerotic lesions. Results Surprisingly, RNA/gDNA parallel sequencing revealed that gene expression profiles of mutant monocytes resembled those of non-mutant monocytes in carriers of DNMT3A-CHIP mutations (n=4). Collectively, however, monocytes of DNMT3A mutation carriers were more inflammatory than those of matched non-carrier patients. Mutant hematopoietic stem and progenitor cells, analyzed by RNA/gDNA parallel sequencing in two carriers, expressed more activation and inflammatory markers compared to non-mutant cells within the same bone marrow, and intraindividual differences declined as cells differentiated into monocytes. Macrophages were isolated from atherosclerotic aortas of mixed bone marrow chimeric mice based on CD45.1 (non-mutant) or CD45.2 (mutant or non-mutant controls) expression and subjected to single cell RNA sequencing. Inflammation markers were higher expressed in non-mutant macrophages within carrier mice than in non-mutant macrophages of non-carrier chimeras, and showed relatively small differences compared to Dnmt3a-deficient macrophages. Conclusions Our experimental findings in humans and mice support a pathomechanistic model in which few DNMT3A-CHIP mutation-carrying hematopoietic stem cells stimulate non-mutant cells within the bone marrow, thereby augmenting inflammation downstream in all circulating monocytes and their progeny in atherosclerotic lesions.

Blood Lipoproteins Shape the Phenotype and Lipid Content of Early Atherosclerotic Lesion Macrophages: A Dual-Structured Mathematical Model

Macrophages in atherosclerotic lesions exhibit a spectrum of behaviours or phenotypes. The phenotypic distribution of monocyte-derived macrophages (MDMs), its correlation with MDM lipid content, and relation to blood lipoprotein densities are not well understood. Of particular interest is the balance between low density lipoproteins (LDL) and high density lipoproteins (HDL), which carry bad and good cholesterol respectively. To address these issues, we have developed a mathematical model for early atherosclerosis in which the MDM population is structured by phenotype and lipid content. The model admits a simpler, closed subsystem whose analysis shows how lesion composition becomes more pathological as the blood density of LDL increases relative to the HDL capacity. We use asymptotic analysis to derive a power-law relationship between MDM phenotype and lipid content at steady-state. This relationship enables us to understand why, for example, lipid-laden MDMs have a more inflammatory phenotype than lipid-poor MDMs when blood LDL lipid density greatly exceeds HDL capacity. We show further that the MDM phenotype distribution always attains a local maximum, while the lipid content distribution may be unimodal, adopt a quasi-uniform profile or decrease monotonically. Pathological lesions exhibit a local maximum in both the phenotype and lipid content MDM distributions, with the maximum at an inflammatory phenotype and near the lipid content capacity respectively. These results illustrate how macrophage heterogeneity arises in early atherosclerosis and provide a framework for future model validation through comparison with single-cell RNA sequencing data.

Abstract 2052: Macrophage-specific Ripk1 Knockdown Reduces Macrophage Inflammation And Atherosclerosis Progression In Ldlr -/- Mice.

Background: Chronic activation of the innate immune system drives inflammation and contributes to atherosclerosis. Pro-inflammatory agents (oxidised LDL, IL-1β) drive lesional inflammation and development, whereas anti-inflammatory agents (IL-10, HDL, Apolipoprotein-AI), promote an anti-inflammatory milieu and resolution. RIPK1 (Receptor interacting protein kinase 1) is a master regulatory gene that decides whether a cell undergoes inflammation, apoptosis and/or necroptosis, depending on its phosphorylation or ubiquitination status. We have previously observed that RIPK1 gene expression is increased in both endothelial cells and macrophages in early atherosclerotic lesions (Karunakaran et al, Circulation 2022). Here, we seek to determine whether macrophage-specific RIPK1 contributes to atherosclerosis. Methods: Given that Ripk1 -/- mice are lethal, we performed bone marrow (BM) transplants of Ripk1 +/- and wild-type (wt) littermate BM into female and male Ldlr -/- mice (n=8-12/grp) and mice were fed a western diet for 12-16 wks. In vitro experiments were performed in BM derived macrophages from wt and Ripk1 +/- mice. Results: Ldlr -/- mice recipient of Ripk1 +/- BM and western diet feeding for 12 or 16 wk had a marked reduction in aortic sinus lesion area (68.9% and 49.7% respectively, p<0.05) compared to their wt controls. Preliminary analysis show that there is a reduction in lesional macrophage area in Ripk1 +/- BM recipient Ldlr -/- mice relative to control (0.02±0.01 vs 0.01±0.006mm, p<0.01), suggesting that macrophage infiltration was reduced. Interestingly, anti-inflammatory agents, including recombinant IL10, IL13 (both 100ng/ml), Apolipoprotein-AI and discoidal HDL (both 500ug/mL) markedly reduced macrophage Ripk1 expression in vitro (by ~50%, p<0.05), likely reducing Ripk1-dependent inflammation and promoting an anti-inflammatory milieu. Current mechanistic studies are investigating the transcriptional regulator, STAT3, as a key regulator of Ripk1 expression. Conclusion: We identified macrophage-RIPK1 as a central driver of atherosclerosis. These timely studies highlight the therapeutic potential of dampening Ripk1 overexpression, as opposed to the detrimental abolishment of Ripk1, in treating atherosclerosis.

Mitochondrial dysfunction and metabolic reprogramming induce macrophage pro-inflammatory phenotype switch and atherosclerosis progression in aging.

Aging increases the risk of atherosclerotic vascular disease and its complications. Macrophages are pivotal in the pathogenesis of vascular aging, driving inflammation and atherosclerosis progression. NOX4 (NADPH oxidase 4) expression increases with age, correlating with mitochondrial dysfunction, inflammation, and atherosclerosis. We hypothesized that the NOX4-dependent mitochondrial oxidative stress promotes aging-associated atherosclerosis progression by causing metabolic dysfunction and inflammatory phenotype switch in macrophages. We studied atherosclerotic lesion morphology and macrophage phenotype in young (5-month-old) and aged (16-month-old) Nox4 -/-/Apoe -/- and Apoe -/- mice fed Western diet. Young Nox4-/-/Apoe-/- and Apoe-/- mice had comparable aortic and brachiocephalic artery atherosclerotic lesion cross-sectional areas. Aged mice showed significantly increased lesion area compared with young mice. Aged Nox4-/-/Apoe-/- had significantly lower lesion areas than Apoe-/- mice. Compared with Apoe-/- mice, atherosclerotic lesions in aged Nox4-/-/Apoe-/- showed reduced cellular and mitochondrial ROS and oxidative DNA damage, lower necrotic core area, higher collagen content, and decreased inflammatory cytokine expression. Immunofluorescence and flow cytometry analysis revealed that aged Apoe-/- mice had a higher percentage of classically activated pro-inflammatory macrophages (CD38+CD80+) in the lesions. Aged Nox4-/-/Apoe-/- mice had a significantly higher proportion of alternatively activated pro-resolving macrophages (EGR2+/CD163+CD206+) in the lesions, with an increased CD38+/EGR2+ cell ratio compared with Apoe-/- mice. Mitochondrial respiration assessment revealed impaired oxidative phosphorylation and increased glycolytic ATP production in macrophages from aged Apoe-/- mice. In contrast, macrophages from Nox4-/-/Apoe-/- mice were less glycolytic and more aerobic, with preserved basal and maximal respiration and mitochondrial ATP production. Macrophages from Nox4-/-/Apoe-/- mice also had lower mitochondrial ROS levels and reduced IL1β secretion; flow cytometry analysis showed fewer CD38+ cells after IFNγ+LPS treatment and more EGR2+ cells after IL4 treatment than in Apoe-/- macrophages. In aged Apoe-/- mice, inhibition of NOX4 activity using GKT137831 significantly reduced macrophage mitochondrial ROS and improved mitochondrial function, resulting in decreased CD68+CD80+ and increased CD163+CD206+ lesion macrophage proportion and attenuated atherosclerosis. Our findings suggest that increased NOX4 in aging drives macrophage mitochondrial dysfunction, glycolytic metabolic switch, and pro-inflammatory phenotype, advancing atherosclerosis. Inhibiting NOX4 or mitochondrial dysfunction could alleviate vascular inflammation and atherosclerosis, preserving plaque integrity.

Therapeutic efficacies of mitochondria-targeted esculetin and metformin in the improvement of age-associated atherosclerosis via regulating AMPK activation.

Atherosclerosis, in general, is an age-associated cardiovascular disease wherein a progressive decline in mitochondrial function due to aging majorly contributes to the disease development. Mitochondria-derived ROS due to dysregulated endothelial cell function accentuates the progression of atherosclerotic plaque formation. To circumvent this, mitochondrially targeted antioxidants are emerging as potential candidates to combat metabolic abnormalities. Recently, we synthesized an alkyl TPP+ tagged esculetin (Mito-Esc), and in the current study, we investigated the therapeutic efficacies of Mito-Esc and metformin, a well-known anti-diabetic drug, in the amelioration of age-associated plaque formation in the aortas of 12months aged Apoe-/- and 20months aged C57BL/6 mice, in comparison to young C57BL/6 control mice. Administration of Mito-Esc or metformin significantly reduced age-induced atherosclerotic lesion area, macrophage polarization, vascular inflammation, and senescence. Further, chronic passaging of human aortic endothelial cells (HAEC) with either Mito-Esc or metformin significantly delayed cellular senescence via the activation of the AMPK-SIRT1/SIRT6 axis. Conversely, depletion of either AMPK/SIRT1/SIRT6 caused premature senescence. Consistent with this, Mito-Esc or metformin treatment attenuated NFkB-mediated inflammatory signaling and enhanced ARE-mediated anti-oxidant responses in comparison to late passage control HAECs. Importantly, culturing of HAECs for several passages with either Mito-Esc or metformin significantly improved mitochondrial function. Overall, Mito-Esc and metformin treatments delay age-associated atherosclerosis by regulating vascular senescence via the activation of AMPK-SIRT1/SIRT6 axis.

Abstract 15270: Macrophage ADAR1 Regulates Atherosclerosis Development Through Editing-Independent Degradation of PPAR-γ

Background: Macrophage activation and vascular inflammation play important roles in atherosclerosis. Non-editing functions of adenosine deaminase acting on RNA (ADAR1) in macrophage activation and atherosclerosis remain elusive. Methods: Nonvascular ADAR1 in atherosclerosis progression was investigated via aortic transplantation and bone marrow transplantation. ApoE-/- mice combined with macrophage-specific ADAR1 deficiency were used to determine macrophage-specific roles of ADAR1 in atherosclerosis. Human coronary atherosclerotic specimens were utilized to establish the relevance to human atherosclerosis. Moreover, a humanized atherosclerosis model was created to examine the influence of human macrophages in human arteries. Results: ADAR1+/– attenuates atherosclerosis. Allograft transplantation of wild-type abdominal aorta into ADAR1+/– recipient mice promoted atherosclerosis. Likewise, bone marrow transplantation from wild-type mice to ADAR1+/– recipient mice negated the protective effects of ADAR1+/–, suggesting that nonvascular ADAR1 is instrumental for atherosclerosis progression. ADAR1 deficiency in hematopoietic cells lessened the prevalence and severity of atherosclerosis, and impeded macrophage infiltration, foam cell formation, and aortic wall inflammation. Mechanistically, ADAR1 deletion deterred classical macrophage activation and foam cell formation via downregulation of PPAR-γ, which was mediated by enhancing ubiquitination and further degradation of PPAR-γ. Importantly, ADAR1 was upregulated in macrophages in human atherosclerotic lesions. Transplantation of ADAR1-deficient human monocytes, rather than their wild-type counterparts, into hyperlipidemic immunodeficient mice suppressed the formation of atherosclerosis in transplanted arteries from patients undergoing coronary artery bypass grafting surgery due to coronary artery blockade by atherosclerotic plagues. Interestingly, ADAR1 suppression in macrophages noticeably augmented the anti-atherosclerotic effect of the PPAR-γ agonist rosiglitazone. Conclusions: These results demonstrate that noncanonical ADAR1 plays an essential role in the regulation of macrophage activation and atherosclerosis development.

Enhanced Akt3 kinase activity reduces atherosclerosis in hyperlipidemic mice in a gender-dependent manner

Akt3 is one of the three members of the serine/threonine protein kinase B (AKT) family, which regulates multiple cellular processes. We have previously demonstrated that global knockout of Akt3 in mice promotes atherogenesis in a macrophage-dependent manner. Whether enhanced Akt3 kinase activity affects atherogenesis is not known. In this study, we crossed atherosclerosis-prone ApoE-/- mice with a mouse strain that has enhanced Akt3 kinase activity (Akt3nmf350) and assessed atherosclerotic lesion formation and the role of macrophages in atherogenesis. Significant reduction in atherosclerotic lesion area and macrophage accumulation in lesions were observed in ApoE-/-/Akt3nmf350 mice fed a Western-type diet. Experiments using chimeric ApoE-/- mice with either ApoE-/-/Akt3nmf350 bone marrow or ApoE-/- bone marrow cells showed that enhanced Akt3 activity specifically in bone marrow-derived cells is atheroprotective. The atheroprotective effect of Akt3nmf350 was more pronounced in male mice. In line with this result, the release of the pro-inflammatory cytokines IL-6, MCP1, TNF-α, and MIP-1α was reduced by macrophages from male but not female ApoE-/-/Akt3nmf350 mice. Levels of IL-6 and TNF-α were also reduced in atherosclerotic lesions of ApoE-/-/Akt3nmf350 male mice compared to ApoE-/- mice. Macrophages from male ApoE-/-/Akt3nmf350 mice were also more resistant to apoptosis invitro and invivo and tended to have more pronounced M2 polarization invitro. These findings demonstrated that enhanced Akt3 kinase activity in macrophages protects mice from atherosclerosis in hyperlipidemic mice in a gender-dependent manner.

Development of LOX-1 Antibody Modified Immuno-liposomes as Drug Carriers to Macrophages in Atherosclerotic Lesions.

We developed a drug delivery system for atherosclerotic lesions using immuno-liposomes. We focused on enhancing the delivery efficiency of the liposomes to macrophages in atherosclerotic lesions by antibody modification of lectinlike oxidized low-density lipoproteins (LDL) receptor 1 (LOX-1). The cellular accumulation of the liposomes in foam cells induced by oxidized LDL (oxLDL) in Raw264 mouse macrophages was evaluated. The cellular accumulation of LOX-1 antibody modified liposomes in oxLDL-induced foam cells and untreated Raw264 cells was significantly higher compared with that of unmodified liposomes. The liposomes were also administered intravenously to Apoeshl mice as an atherosclerosis model. Frozen sections were prepared from the mouse aortas and observed by confocal laser microscopy. The distribution of LOX-1 antibody modified liposomes in the atherosclerotic regions of Apoeshl mice was significantly greater compared with that of unmodified liposomes. The results suggest that LOX-1 antibody modified liposomes can target foam cells in atherosclerotic lesions, providing a potential route for delivering various drugs with pharmacological effects or detecting atherosclerotic foci for the diagnosis of atherosclerosis.

A novel cuproptosis-related diagnostic gene signature and differential expression validation in atherosclerosis

Atherosclerosis (AS) is a major contributor to morbidity and mortality worldwide. However, the molecular mechanisms and mediator molecules involved remain largely unknown. Copper, which plays an essential role in cardiovascular disease, has been suggested as a potential risk factor. Copper homeostasis is closely related to the occurrence and development of AS. Recently, a new cell death pathway called cuproptosis has been discovered, which is driven by intracellular copper excess. However, no previous studies have reported a relationship between cuproptosis and AS. In this study, we integrated bulk and single-cell sequencing data to screen and identify key cuproptosis-related genes in AS. We used correlation analysis, enrichment analysis, random forest, and other bioinformatics methods to reveal their relationships. Our findings report, for the first time, the involvement of cuproptosis-related genes FDX1, SLC31A1, and GLS in atherogenesis. FDX1 and SLC31A1 were upregulated, while GLS was downregulated in atherosclerotic plaque. Receiver operating characteristic curves demonstrate their potential diagnostic value for AS. Additionally, we confirm that GLS is mainly expressed in vascular smooth muscle cells, and SLC31A1 is mainly localized in macrophages of atherosclerotic lesions in experiments. These findings shed light on the cuproptosis landscape and potential diagnostic biomarkers for AS, providing further evidence about the vital role of cuproptosis in atherosclerosis progression.

458-P: Proinflammatory Macrophage Phenotype Modulates Vascular Smooth Muscle Cell Differentiation via the Histone Demethylase JMJD3

Patients with diabetes are at increased risk of a multitude of vascular diseases, including atherosclerosis, which is driven by macrophage-mediated inflammation. While it is known that atherosclerosis is characterized by a proliferative, synthetic smooth muscle phenotype that underlies disease, the exact role of other cell types, namely myeloid cells and their interaction with smooth muscle cells is less understood. Furthermore, how diabetes modifies atherosclerotic severity is another important question. To address this, we performed an epigenetic superarray to identify key chromatin modifying enzymes in vascular smooth muscle cells (vSMC) from normal diet (ND) and diabetic-induced obese (DIO) aortas. We identified the histone demethylase, JMJD3, which was significantly decreased in DIO compared to ND vSMC (p<0.05). Furthermore, in our single cell RNA-seq data set from human atherosclerotic lesions, JMJD3 was expressed in smooth muscle cells. It is known that macrophages in atherosclerotic lesions exhibit a pro-inflammatory phenotype. To model this pro-inflammatory process, we stimulated bone marrow derived macrophages with LPS and found that supernatant from LPS-stimulated BMDMs significantly inhibited smooth muscle specific gene expression (SMA, SM22, CNN1, SM-MHC) as well as expression of JMJD3 compared to control media (p<0.05). Additionally, siRNA-mediated knockdown of JMJD3 in vSMC reduced SMC-specific gene expression, and treatment of mouse aortic vSMC with the JMJD3 selective inhibitor, GSK4J, decreased smooth muscle gene expression (p<0.05). Our data shows that pro-inflammatory macrophages drive reduced vascular smooth muscle gene expression and that JMJD3 in SMC is a regulator of this process. These results implicate JMJD3 in macrophage-smooth muscle cell crosstalk in the pro-inflammatory state and identify it as a potential therapeutic target in the management of atherosclerosis. Disclosure K.D.Mangum: None. A.Joshi: None. P.Mazumder: None. S.Wolf-fortune: None. E.Barrett: None. B.Moore: None. F.M.Davis: None. K.Gallagher: None. Funding National Institutes of Health (1F32DK131799-01); American College of Surgeons; Vascular & Endovascular Surgery Society

Cholesterol-load evokes robust calcium response in macrophages: An early event toward cholesterol-induced macrophage death

Macrophages in atherosclerotic lesions accumulate large amounts of unesterified cholesterol. Excess cholesterol load leads to cell death of macrophages, which is associated with the progression of atherosclerotic lesions. Calcium depletion in the endoplasmic reticulum (ER) and subsequent pro-apoptotic aberrant calcium signaling are key events in cholesterol-induced macrophage death. Although these concepts imply cytoplasmic calcium events in cholesterol-loaded macrophages, the mechanisms linking cholesterol accumulation to cytoplasmic calcium response have been poorly investigated. Based on our previous finding that extracellularly applied cholesterol evoked robust calcium oscillations in astrocytes, a type of glial cells in the brain, we hypothesized that cholesterol accumulation in macrophages triggers cytoplasmic calcium elevation. Here, we showed that cholesterol application induces calcium transients in THP-1-derived and peritoneal macrophages. Inhibition of inositol 1,4,5-trisphosphate receptors (IP3Rs) and l-type calcium channels (LTCCs) prevented cholesterol-induced calcium transients and ameliorated cholesterol-induced macrophage death. These results suggest that cholesterol-induced calcium transients through IP3Rs and LTCCs are crucial mechanisms underlying cholesterol-induced cell death of macrophages.

Macrophage DCLK1 promotes atherosclerosis via binding to IKKβ and inducing inflammatory responses

Atherosclerosis is a chronic inflammatory disease with high morbidity and mortality rates worldwide. Doublecortin‐like kinase 1 (DCLK1), a microtubule‐associated protein kinase, is involved in neurogenesis and human cancers. However, the role of DCLK1 in atherosclerosis remains undefined. In this study, we identified upregulated DCLK1 in macrophages in atherosclerotic lesions of ApoE−/− mice fed an HFD and determined that macrophage‐specific DCLK1 deletion attenuates atherosclerosis by reducing inflammation in mice. Mechanistically, RNA sequencing analysis indicated that DCLK1 mediates oxLDL‐induced inflammation via NF‐κB signaling pathway in primary macrophages. Coimmunoprecipitation followed by LC–MS/MS analysis identified IKKβ as a binding protein of DCLK1. We confirmed that DCLK1 directly interacts with IKKβ and phosphorylates IKKβ at S177/181, thereby facilitating subsequent NF‐κB activation and inflammatory gene expression in macrophages. Finally, a pharmacological inhibitor of DCLK1 prevents atherosclerotic progression and inflammation both in vitro and in vivo. Our findings demonstrated that macrophage DCLK1 promotes inflammatory atherosclerosis by binding to IKKβ and activating IKKβ/NF‐κB. This study reports DCLK1 as a new IKKβ regulator in inflammation and a potential therapeutic target for inflammatory atherosclerosis.

Identification of candidate biomarkers and mechanisms in foam cell formation from heterogeneous cellular origins via integrated transcriptome analysis.

Atherosclerosis is an underlying cause of cardiovascular disease which is a leading cause of death worldwide. Foam cells play a crucial role in atherosclerotic lesion development, and macrophages and vascular smooth muscle cells (VSMCs) appear to contribute to the formation of the majority of atheromatous foam cells via oxidized low-density lipoprotein (ox-LDL) uptake. An integrated, microarray-based analysis using GSE54666 and GSE68021, which contain samples of human macrophages and VSMCs incubated with ox-LDL, was conducted. The differentially expressed genes (DEGs) in each dataset were investigated via the linear models for microarray data (limma) v. 3.40.6 software package in R v. 4.1.2 (The R Foundation for Statistical Computing). Gene ontology (GO) and pathway enrichment were performed via the ClueGO v. 2.5.8 and CluePedia v. 1.5.8 databases and the Database of Annotation, Visualization and Integrated (DAVID; https://david.ncifcrf.gov). The convergent DEGs in the two cell types were obtained, and the protein interactions and network of transcriptional factors were analyzed using the Search Tool for the Retrieval of Interacting Genes (STRING) v. 11.5 and the Transcriptional Regulatory Relationships Unraveled by Sentence-based Text-mining (TRRUST) v. 2 databases. The selected DEGs were further validated using external data from GSE9874, and a machine learning algorithm of the least absolute shrinkage and selection operator (LASSO) regression and receiver operating characteristic (ROC) analysis were applied to explore the candidate biomarkers. We discovered the significant DEGs and pathways that were shared or unique among the 2 cell types, coupling with enriched lipid metabolism in macrophages, and upregulated defense response in VSMCs. Moreover, we identified BTG2, ABCA1, and SLC7A11 as potential biomarkers and molecular targets for atherogenesis. Our study provides a comprehensive summary of the landscape of the transcriptional regulations in macrophages and VSMCs under ox-LDL treatment from a bioinformatics perspective, which may contribute to a better understanding of the pathophysiological mechanisms of foam cell formation.

Comprehensive Lipid Profiling Recapitulates Enhanced Lipolysis and Fatty Acid Metabolism in Intimal Foamy Macrophages From Murine Atherosclerotic Aorta.

Lipid accumulation in macrophages is a prominent phenomenon observed in atherosclerosis. Previously, intimal foamy macrophages (FM) showed decreased inflammatory gene expression compared to intimal non-foamy macrophages (NFM). Since reprogramming of lipid metabolism in macrophages affects immunological functions, lipid profiling of intimal macrophages appears to be important for understanding the phenotypic changes of macrophages in atherosclerotic lesions. While lipidomic analysis has been performed in atherosclerotic aortic tissues and cultured macrophages, direct lipid profiling has not been performed in primary aortic macrophages from atherosclerotic aortas. We utilized nanoflow ultrahigh-performance liquid chromatography-tandem mass spectrometry to provide comprehensive lipid profiles of intimal non-foamy and foamy macrophages and adventitial macrophages from Ldlr-/- mouse aortas. We also analyzed the gene expression of each macrophage type related to lipid metabolism. FM showed increased levels of fatty acids, cholesterol esters, phosphatidylcholine, lysophosphatidylcholine, phosphatidylinositol, and sphingomyelin. However, phosphatidylethanolamine, phosphatidic acid, and ceramide levels were decreased in FM compared to those in NFM. Interestingly, FM showed decreased triacylglycerol (TG) levels. Expressions of lipolysis-related genes including Pnpla2 and Lpl were markedly increased but expressions of Lpin2 and Dgat1 related to TG synthesis were decreased in FM. Analysis of transcriptome and lipidome data revealed differences in the regulation of each lipid metabolic pathway in aortic macrophages. These comprehensive lipidomic data could clarify the phenotypes of macrophages in the atherosclerotic aorta.

Differential Expression of Inflammarafts in Macrophage Foam Cells and in Nonfoamy Macrophages in Atherosclerotic Lesions-Brief Report.

Reprogramming of monocytes and macrophage manifests in hyperinflammatory responses and chronification of inflammation in atherosclerosis. Recent studies focused on epigenetic, transcriptional, and metabolic alterations that characterize trained immunity. However, the underlying effector mechanisms driving the hyperinflammatory response of reprogrammed macrophages remain unclear. We hypothesized that the plasma membrane of atherosclerotic lesion macrophages undergoes reprogramming to maintain inflammarafts, enlarged lipid rafts (LR) serving as a platform for assembly of inflammatory receptor complexes. Single-cell suspensions from the aortae of Western diet-fed Ldlr-/- mice were gated for BODIPY-high foamy and BODIPY-low nonfoamy F4/80 macrophages by flow cytometry. Inflammarafts were characterized by increased levels of LR, TLR4 (toll-like receptor-4) localization to LR, TLR4 dimers, and the proximity between TLR2, TLR1, and CD36. In a cellular model of trained immunity, LR, TLR4 dimers, and the inflammatory response were measured in bone marrow-derived macrophages subjected to a 24-hour treatment with LPS (lipopolysaccharide) or OxLDL (oxidized low-density lipoprotein), followed by a 6-day wash-out period. Nonfoamy macrophages, which constituted ≈40% of macrophages in atherosclerotic lesions, expressed significantly higher levels of LR and TLR4 dimers, as well as proximity ligation signals for TLR4-LR, TLR2-CD36, and TLR2-TLR1 complexes, compared with foamy macrophages. These inflammaraft measures associated, to a different degree, with plasma cholesterol and inflammatory cytokines, as well as the size of the atherosclerotic lesions and necrotic cores. The bone marrow-derived macrophages trained with LPS simulated nonfoamy atherosclerotic lesion macrophages and continued to express inflammarafts and inflammatory genes for 6 days after LPS removal and displayed a hyperinflammatory response to Pam3CSK4, a TLR2/TLR1 agonist. OxLDL-exposed, lipid-laden macrophages did not express inflammarafts. Our data support the hypothesis that persistent inflammarafts in nonfoamy macrophages in atherosclerotic lesions serve as effectors of macrophage reprogramming into a hyperinflammatory phenotype.

Abstract 13518: Augmenting SOD2 Levels in Apoe -/- Mice With Peptide-mRNA Nanoparticles Preserves Atherosclerotic Plaque Stability in Advanced Atherosclerosis

Erosion or rupture of the advanced atherosclerotic plaque is the leading cause of atherosclerosis complications such as acute coronary syndrome and stroke. Atherosclerotic plaques in aging hypercholesterolemic mice have decreased SOD2 levels and increased mitochondrial oxidative stress, which leads to cell apoptosis, necrotic core expansion, and fibrous cap thinning and rupture. We hypothesized that increasing SOD2 expression in advanced atherosclerotic plaque cells in mice using mRNA polyplex would protect mitochondria from oxidative damage and preserve plaque stability. Macrophages isolated from Sod2 +/- mice treated with amphipathic cationic peptide p5RHH: Sod2 mRNA polyplexes had markedly increased levels of Sod2 mRNA and mitochondrial SOD2 (p<0.01), higher SOD activity, which resulted in lower levels of mitochondrial and cellular reactive oxygen species (ROS) and improved mitochondrial function. RNA-Seq analysis showed that macrophages treated with Sod2 nanoplexes had broadly decreased inflammatory cytokines and immune activation pathways. In Apoe -/- mice, atherosclerotic lesions with vulnerable plaque features were induced in the carotid artery by placing a perivascular collar over the carotid artery and 4-months Western diet. Mice were treated for 4 weeks with saline or with 0.5 mg/kg p5RHH: Sod2 mRNA polyplexes which selectively accumulated in atherosclerotic lesion macrophages. The carotid plaques of the mice treated with Sod2 mRNA differed significantly from the plaques of the control Apoe -/- mice by higher SOD2 expression, lower mitochondrial ROS and oxidative DNA damage, and reduced expression of CCL2, IL1, IL6, MMP 2 and 9 (p<0.01). Sod2 polyplexes-treated mouse plaques exhibited 25% fewer inflammatory macrophages (p<0.01) and 98% more smooth muscle cells (p<0.001) with a reduced lipid core, preserved fibromuscular cap, and decreased plaque vulnerability index (p<0.05). The results suggest that selective overexpression of Sod2 in plaque macrophages using nanoplexes reduces mitochondrial stress and preserves plaque stability without adverse effects. Using nanoparticle-based mRNA therapeutics to modulate plaque morphology has great potential in the prevention and treatment of atherosclerosis complications.

Identifying potential signatures for atherosclerosis in the context of predictive, preventive, and personalized medicine using integrative bioinformatics approaches and machine-learning strategies.

Atherosclerosis is a major contributor to morbidity and mortality worldwide. Although several molecular markers associated with atherosclerosis have been developed in recent years, the lack of robust evidence hinders their clinical applications. For these reasons, identification of novel and robust biomarkers will directly contribute to atherosclerosis management in the context of predictive, preventive, and personalized medicine (PPPM). This integrative analysis aimed to identify critical genetic markers of atherosclerosis and further explore the underlying molecular immune mechanism attributing to the altered biomarkers. Gene Expression Omnibus (GEO) series datasets were downloaded from GEO. Firstly, differential expression analysis and functional analysis were conducted. Multiple machine-learning strategies were then employed to screen and determine key genetic markers, and receiver operating characteristic (ROC) analysis was used to assess diagnostic value. Subsequently, cell-type identification by estimating relative subsets of RNA transcript (CIBERSORT) and a single-cell RNA sequencing (scRNA-seq) data were performed to explore relationships between signatures and immune cells. Lastly, we validated the biomarkers' expression in human and mice experiments. A total of 611 overlapping differentially expressed genes (DEGs) included 361 upregulated and 250 downregulated genes. Based on the enrichment analysis, DEGs were mapped in terms related to immune cell involvements, immune activating process, and inflaming signals. After using multiple machine-learning strategies, dehydrogenase/reductase 9 (DHRS9) and protein tyrosine phosphatase receptor type J (PTPRJ) were identified as critical biomarkers and presented their high diagnostic accuracy for atherosclerosis. From CIBERSORT analysis, both DHRS9 and PTPRJ were significantly related to diverse immune cells, such as macrophages and mast cells. Further scRNA-seq analysis indicated DHRS9 was specifically upregulated in macrophages of atherosclerotic lesions, which was confirmed in atherosclerotic patients and mice. Our findings are the first to report the involvement of DHRS9 in the atherogenesis, and the proatherogenic effect of DHRS9 is mediated by immune mechanism. In addition, we confirm that DHRS9 is localized in macrophages within atherosclerotic plaques. Therefore, upregulated DHRS9 could be a novel potential target for the future predictive diagnostics, targeted prevention, patient stratification, and personalization of medical services in atherosclerosis. The online version contains supplementary material available at 10.1007/s13167-022-00289-y.

Roles of OLR1 and IL17A variants on clinical phenotypes of Turkish patients undergoing coronary artery bypass surgery

Abstract Objectives Coronary artery disease (CAD) is a pathological condition resulting from atherosclerosis in the coronary arteries. IL17A has been shown to recruit and activate macrophages in atherosclerotic lesions, thereby participating in plaque destabilization. Currently, whether OLR1 and IL17A variants are involved in the pathogenesis of CAD is unclear. This case-control study aimed to investigate their roles in CAD etiology and prognosis. Methods In this study, 100 severe CAD patients who had undergone the coronary artery bypass graft surgery and 100 healthy controls were genotyped for OLR1 rs11053646, IL17A rs3819025, and rs8193037 variants via RT-PCR. Results The patients with OLR1 rs11053646 CG + GG genotype demonstrated a higher frequency of multi-vessel stenosis (18%) than single- (11.10%) or double-vessel (13.30%) stenosis (p=0.77). Additionally, although not statistically significant, this group of patients had 6.280 times more CAD risk than CC genotype carriers (p=0.089). Furthermore, logistic regression analysis revealed significant associations between the three variants and the risk factors for CAD development, namely waist circumference (p=0.002), body mass index (p=0.013), fasting glucose level (p=0.006), and triglyceride levels (p=0.035). Conclusions OLR1 rs11053646, IL17A rs3819025, and rs8193037 variants do not increase the risk for CAD development. However, this conclusion should be confirmed with a larger cohort.

Abstract 451: Enrichment Of High Density Lipoprotein-Associated Protease Inhibitor Activity Prevents Atherosclerosis Progression

Background: Atherosclerotic lesions contain inflammatory cells which secrete a variety of proteolytic enzymes that contribute to lesion progression. The relative contributions of specific proteases to lesion progression is not well understood. The serine protease elastase is produced by neutrophils and macrophages in atherosclerotic lesions and may contribute to plaque progression and destabilization. We have reported significant enrichment of protease inhibitor proteins associated with plasma high density lipoprotein (HDL), including the elastase inhibitor alpha-1-antitrypsin, suggesting a potential role for HDL as an endogenous modulator of protease activity. Hypothesis: We hypothesized that protease inhibition is a mechanism for HDL-mediated protection against atherosclerosis that is independent of HDL cholesterol content. Methods and Results: We developed an HDL-targeting protease inhibitor (HTPI) peptide to allow for synthetic enrichment of HDL-associated elastase inhibitor activity. The small (1 kDa) peptide has an HDL-targeting domain, a soluble linker, and an elastase inhibitor motif. Using a fluorescent version, we demonstrated preferential binding to HDL in human plasma ex vivo . When injected into mice, we observed binding to plasma HDL and increased elastase inhibitor activity on HDL isolated from plasma of mice that received HTPI. Injection into mouse models of atherosclerosis resulted in accumulation of HTPI within lesions. To examine the impact of HTPI treatment on established atherosclerosis, Ldlr -/- mice were maintained on Western diet for 12 weeks, then administered vehicle or HTPI (2.5 mg/kg) three times per week for two weeks while remaining on diet. Lesion quantification by en face analysis and aortic root sections demonstrated that HTPI prevented additional lipid deposition in established lesions. The impact of HTPI on lesion morphology and inflammatory features was examined by histology and immunofluorescence staining of aortic root sections. Conclusions: Targeted enrichment of HDL-associated elastase inhibitor activity using a synthetic peptide prevented atherosclerotic lesion progression. These findings support a possible role for the endogenous antiprotease activity of HDL in atheroprotection.

MiR-223-3p inhibits the progression of atherosclerosis via down-regulating the activation of MEK1/ERK1/2 in macrophages.

Background: microRNAs (miRNAs) have drawn more attention to the progression of atherosclerosis (AS), due to their noticeable inflammation function in cardiovascular disease. Macrophages play a crucial role in disrupting atherosclerotic plaque, thereby we explored the involvement of miR-223-3p in the inflammatory response in macrophages.Methods: RT-qPCR was used to analyze the miR-223-3p levels in carotid arteries and serum of AS patients. ROC curve was used to assess the diagnostic value of miR-223-3p. Movat staining was applied to evaluate the morphological differences. FISH was used to identify the expression of miR-223-3p in macrophages of atherosclerotic lesions. Bioinformatic analysis was performed. Double-immunofluorescence and western blot were performed to assess the inflammatory cytokine secretion and p-ERK1/2. C16-PAF was injected into the culture medium of the miR-223-3p mimic/NC-transfected macrophages with ox-LDL.Results: MiR-223-3p was up-regulated in AS patients and was associated with a higher overall survival rate. MiR-223-3p was co-localized with CD68+ macrophages in vulnerable atherosclerotic lesions. MiR-223-3p mimics decreased atherosclerotic lesions, macrophages numbers whereas increased SMCs numbers in the lesions. The TNF-a immune-positive areas were reduced by miR-223-3p mimics. MAP2K1 was negatively associated with miR-223-3p. MiR-223-3p mimics reduced the inflammation and the MEK1/ERK1/2 signaling pathway in vivo and in vitro. C16-PAF reversed the effects of miR-223-3p mimics on inflammation and ERK1/2 signaling pathway.Conclusions: MiR-223-3p negatively regulates inflammatory responses by the MEK1/ERK1/2 signaling pathway. Our study provides new insight into how miR-223-3p protects against atherosclerosis, representing a broader therapeutic prospect for treating atherosclerosis by miR-223-3p.