Atherosclerotic Aorta Research Articles

Extracellular peroxiredoxin 5 exacerbates atherosclerosis via the TLR4/MyD88 pathway

BACKGROUNGD and AIMSPeroxiredoxin 5 (PRDX5), an atypical 2-Cys peroxiredoxin (PRDX), is known to regulate a global oxidative stresses and inflammatory responses. Inflammation and oxidative stress are pivotal factors in the development of atherosclerosis, especially in the context of vascular endothelial dysfunction. However, effects of PRDX5 on the atherosclerosis remain unclear. This study aimed to elucidate the role of PRDX5 in the pathogenesis of atherosclerosis. METHODSFor in vivo analysis, normal chow dieted 60-weeks old Apolipoprotein E knockout (ApoE-/-) and Prdx5-/-; ApoE-/- mice were used for experiments. For In vitro, human umbilical vein endothelial cells (HUVECs) were stimulated with oxidized LDL (oxLDL; 50ng/ml) for 24hrs following serum starvation by incubating with serum-free Endothelial Cell Growth Medium-2 (EGM-2) for 1hr. RESULTSWe observed elevated PRDX5 expression under atherosclerotic conditions in both humans and mice. Unexpectedly, Prdx5-/-; ApoE-/- mice exhibited reduced plaque formation, with no discernible difference in aortic hydrogen peroxide (H2O2) levels compared to ApoE-/- mice. Additionally, there was a notable decrease in macrophage accumulation and vascular inflammation in the atherosclerotic aorta of Prdx5-/-; ApoE-/-. For In vitro analysis, HUVECs stimulated with oxLDL showed upregulated PRDX5 expression in both lysate and culture medium. Moreover, PRDX5 knockdown in oxLDL-stimulated (oxLDL-siPRDX5) HUVECs significantly reduced the migration and adhesion of the human monocytic cells (THP-1) to HUVECs, indicating diminished vascular immune responses. Mechanistically, both in vivo and In vitro, PRDX5 deficiency inhibited the Toll-like receptor 4 (TLR4)/Myeloid differentiation primary response 88 (MyD88) signaling pathway, resulting in reduced nuclear factor kappa B (NF-κB) and P38 phosphorylation. Furthermore, treatment with recombinant PRDX5 (rPRDX5) protein restored TLR4/MyD88 signaling in oxLDL-siPRDX5 HUVECs. CONCLUSIONSThese data demonstrate that extracellular PRDX5 contributes to endothelial inflammation, promoting macrophage accumulation in the atherosclerotic aorta through activating TLR4/MyD88/NF-κB and P38 signaling pathways, thereby exacerbating the progression of atherosclerosis.

Activation of receptor-independent fluid-phase pinocytosis promotes foamy monocyte formation in atherosclerotic mice

Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of death worldwide. Clinical and experimental data demonstrated that circulating monocytes internalize plasma lipoproteins and become lipid-laden foamy cells in hypercholesterolemic subjects. This study was designed to identify the endocytic mechanisms responsible for foamy monocyte formation, perform functional and transcriptomic analysis of foamy and non-foamy monocytes relevant to ASCVD, and characterize specific monocyte subsets isolated from the circulation of normocholesterolemic controls and hypercholesterolemic patients. We hypothesized that activation of fluid-phase macropinocytosis contributes to foamy monocyte formation in vitro and in hypercholesterolemic mice in vivo. High resolution scanning electron microscopy (SEM) and quantification of FITC/TRITC-dextran internalization demonstrated macropinocytosis stimulation in human (THP-1) and wild type murine monocytes. Stimulation of macropinocytosis induced foamy monocyte formation in the presence of unmodified, native LDL (nLDL) and oxidized LDL (ox-LDL) in vitro. Genetic blockade of macropinocytosis (LysMCre+Nhe1f/f) inhibited foamy monocyte formation in hypercholesterolemic mice in vivo and attenuated monocyte adhesion to atherosclerotic aortas ex vivo. Mechanistic studies identified NADPH oxidase 2 (Nox2)-derived superoxide (O2⋅−) as an important downstream signaling molecule stimulating macropinocytosis in monocytes. qRT-PCR identified CD36 as a major SR that increases in response to lipid loading in monocytes and deletion of CD36 (Cd36-/-) inhibited foamy monocyte formation in hypercholesterolemic mice. Bulk RNA-sequencing characterized transcriptional differences between non-foamy and foamy monocytes versus macrophages. Finally, flow cytometry analysis of CD14 and CD16 expression demonstrated a significant increase in intermediate monocytes in hypercholesterolemic patients compared to normocholesterolemic controls. These results provide novel insights into the mechanisms of foamy monocyte formation and potentially identify new therapeutic targets for the treatment of atherosclerosis.

Expression of CCL2 signaling pathway genes in patients with periodontitis and atherosclerosis.

Periodontitis and atherosclerosis are chronic inflammatory diseases characterized by leukocyte infiltration. We investigated the expression of CCL4, CCR5, c-Jun, c-Fos, NF-κB, and CCL2 as well as the possible mechanism involved in the regulation of CCL2 in human periodontitis tissues and atherosclerotic aorta based on previous research on the CCL4/CCR5/c-Jun and c-Fos/CCL2 pathway leading to CCL2 expression in collagen-induced arthritis (CIA) rat. Sixty-five volunteers were recruited and the condition of their gingiva and coronary arteries were assessed. The subjects were divided into four groups: healthy control, chronic periodontitis (CP), coronary artery diseases (CAD), and noncoronary artery diseases (non-CAD). Total RNA was isolated from gingiva in periodontitis patients and control populations and from the aorta in patients with and without CAD. PCR was used to examine CCL4, CCR5, c-Jun, c-Fos, NF-κB, and CCL2 levels. The production of CCL2 in the gingiva and aorta was analyzed by immunostaining. PCR revealed that CCL4, CCR5, and CCL2 mRNA levels were increased in CP patients' gingivae and aortas from coronary artery bypass grafting (CABG) patients. Marked c-Jun, c-Fos, and NF-κB gene productions were detected in CP patients' gingivae but did not show statistical differences between the CAD and non-CAD groups. Stronger immunoreactivity against CCL2 was observed in periodontitis gingiva and aorta from CABG patients. Our findings suggest that the CCL4/CCR5/c-Jun and c-Fos/CCL2 pathways may be involved in CCL2 expression in periodontitis. CCL4, CCR5, and CCL2 might act as possible nodes to link the presence of periodontitis and atherosclerosis.

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.

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 Tu015: Angiopoietin-like 4 Stabilizes Atherosclerotic Plaques by Modulating the Phenotypic Transition of Endothelial Cells and Vascular Smooth Muscle Cells

Atherosclerosis, the leading cause of death, is a vascular disease of chronic inflammation. We recently showed that angiopoietin-like 4 (ANGPTL4) promotes cardiac repair by suppressing pathological inflammation. Given the fundamental contribution of inflammation to atherosclerosis, we assessed the role of ANGPTL4 in the development of atherosclerosis and determined whether ANGPTL4 regulates atherosclerotic plaque stability. We injected ANGPTL4 protein twice a week into atherosclerotic Apoe-/- mice and analyzed the atherosclerotic lesion size, endothelial dysfunction, inflammation, and plaque stability. In atherosclerotic mice, ANGPTL4 reduced the atherosclerotic plaque size, endothelial dysfunction, and vascular inflammation. Furthermore, ANGPTL4 was associated with maintenance of vascular integrity and attenuated endothelial-to-mesenchymal transition (EndMT) in both HUVEC and atherosclerotic plaque of Apoe-/- mice. In the atherosclerotic lesions and fibrous caps, the number of α-SMA(+), SM22α(+), and SM-MHC(+) cells was higher, while the number of CD68(+) and Mac2(+) cells was lower in the ANGPTL4 group. Most importantly, the fibrous cap was significantly thicker in the ANGPTL4 group than in the PBS group. Smooth muscle cells (SMCs) isolated from atherosclerotic aortas showed significantly increased expression of CD68 and Krüppel-like factor 4 (KLF4), a modulator of the vascular SMC phenotype, along with downregulation of α-SMA, and these changes were attenuated by ANGPTL4 treatment. Furthermore, ANGPTL4 reduced TNFa-induced NADPH oxidase 1 (NOX1), a major source of reactive oxygen species, resulting in attenuation of KLF4-mediated SMC phenotypic changes. Our results reveal that ANGPTL4 treatment inhibits atherogenesis and suggest that targeting vascular stability and inflammation may serve as a novel therapeutic strategy to prevent and treat atherosclerosis. Even more importantly, ANGPTL4 treatment inhibited the phenotypic changes of SMCs into macrophage-like cells by downregulating NOX1 activation of KLF4, leading to the formation of more stable plaques.

7-Ketocholesterol promotes cholesterol uptake and inflammation in macrophage: Potential mechanisms of dietary COP in atherosclerosis promotion

This study investigated the effects and mechanisms of 7-ketocholesterol in promoting macrophage foaming and inflammation. Single-cell RNA sequencing showed that dietary cholesterol oxidation products increased the inflammatory macrophage and Trem2high macrophage numbers in the atherosclerotic aorta of ApoE−/− mice. Enrichment analysis demonstrated activation of inflammation-related pathways (NF-κB pathway and NLRP3 inflammasome) in inflammatory macrophages and cholesterol metabolism-related pathways (LDLR and ABCG1) in Trem2high macrophages. An oxidized-LDL-treated macrophage model was employed to verify the effects and mechanisms of 7-KC on macrophage inflammation and foam cell formation. 7-KC promoted the inflammation and M1 polarization of macrophage in association with the activations of the NF-κB pathway and NLRP3 inflammasome. Furthermore, 7-KC-mediated excessive cholesterol accumulation was attributed to enhanced cholesterol uptake rather than inhibition of cholesterol efflux. Consequently, LDLR expression was up-regulated by 7-KC, while no significant effect on ABCG1 expression was observed.

Macrophage-derived FGFR1 drives atherosclerosis through PLCγ-mediated activation of NF-κB inflammatory signalling pathway.

Atherosclerosis (AS) is a leading cause of cardiovascular morbidity and mortality. Atherosclerotic lesions show increased levels of proteins associated with the fibroblast growth factor receptor (FGFR) pathway. However, the functional significance and mechanisms governed by FGFR signalling in AS are not known. In the present study, we investigated fibroblast growth factor receptor 1 (FGFR1) signalling in AS development and progression. Examination of human atherosclerotic lesions and aortas of Apoe-/- mice fed a high-fat diet (HFD) showed increased levels of FGFR1 in macrophages. We deleted myeloid-expressed Fgfr1 in Apoe-/- mice and showed that Fgfr1 deficiency reduces atherosclerotic lesions and lipid accumulations in both male and female mice upon HFD feeding. These protective effects of myeloid Fgfr1 deficiency were also observed when mice with intact FGFR1 were treated with FGFR inhibitor AZD4547. To understand the mechanistic basis of this protection, we harvested macrophages from mice and show that FGFR1 is required for macrophage inflammatory responses and uptake of oxidized LDL. RNA sequencing showed that FGFR1 activity is mediated through phospholipase-C-gamma (PLCγ) and the activation of nuclear factor-κB (NF-κB) but is independent of FGFR substrate 2. Our study provides evidence of a new FGFR1-PLCγ-NF-κB axis in macrophages in inflammatory AS, supporting FGFR1 as a potentially therapeutic target for AS-related diseases.

Activation of Nrf2 inhibits atherosclerosis in ApoE−/− mice through suppressing endothelial cell inflammation and lipid peroxidation

BackgroundNuclear erythroid 2-related factor 2 (Nrf2), a transcription factor, is critically involved in the regulation of oxidative stress and inflammation. However, the role of endothelial Nrf2 in atherogenesis has yet to be defined. In addition, how endothelial Nrf2 is activated and whether Nrf2 can be targeted for the prevention and treatment of atherosclerosis is not explored. MethodsRNA-sequencing and single-cell RNA sequencing analysis of mouse atherosclerotic aortas were used to identify the differentially expressed genes. In vivo endothelial cell (EC)-specific activation of Nrf2 was achieved by injecting adeno-associated viruses into ApoE−/− mice, while EC-specific knockdown of Nrf2 was generated in Cdh5CreCas9floxed-stopApoE−/− mice. ResultsEndothelial inflammation appeared as early as on day 3 after feeding of a high cholesterol diet (HCD) in ApoE−/− mice, as reflected by mRNA levels, immunostaining and global mRNA profiling, while the immunosignal of the end-product of lipid peroxidation (LPO), 4-hydroxynonenal (4-HNE), started to increase on day 10. TNF-α, 4-HNE, and erastin (LPO inducer), activated Nrf2 signaling in human ECs by increasing the mRNA and protein expression of Nrf2 target genes. Knockdown of endothelial Nrf2 resulted in augmented endothelial inflammation and LPO, and accelerated atherosclerosis in Cdh5CreCas9floxed-stopApoE−/− mice. By contrast, both EC-specific and pharmacological activation of Nrf2 inhibited endothelial inflammation, LPO, and atherogenesis. ConclusionsUpon HCD feeding in ApoE−/− mice, endothelial inflammation is an earliest event, followed by the appearance of LPO. EC-specific activation of Nrf2 inhibits atherosclerosis while EC-specific knockdown of Nrf2 results in the opposite effect. Pharmacological activators of endothelial Nrf2 may represent a novel therapeutic strategy for the treatment of atherosclerosis.

Rapamycin reduces atherosclerotic plaque inflammation in aged mice

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): 1. ERA-CVD. Beat ATHERO consortium. 2. Dutch Heart Foundation Aims Aging is the most dominant risk factor of atherosclerotic cardiovascular disease (CVD), the main underlying cause of death worldwide. Both aging and atherosclerosis are associated with changes in the composition and function of the immune system, such as an enlarged T cell effector memory pool and elevated pro-inflammatory cytokine secretion. Since the accumulation of these pro-inflammatory factors contribute to atherosclerosis progression, halting inflammation might be a promising strategy to combat CVD. Rapamycin is an immunosuppressant that was recently discovered to increase lifespan due to the inhibition of senescent cells and effector T cells. We therefore hypothesized that rapamycin treatment can attenuate pro-atherogenic immunity in aged mice. Methods To explore the effects of rapamycin treatment, we administrated 1 mg/kg rapamycin or control solvent to aged (22 months old) male atherosclerotic Ldlr-/- mice (n=16 per group) three times a week intraperitoneally. After eight weeks, mice were sacrificed and their organs were collected for flow cytometry, single-cell RNA sequencing and histological analysis. Results Using flow cytometry, we revealed that rapamycin treatment decreased total T cell numbers in the atherosclerotic aorta and spleen compared to control treatment and attributed to a 24% increase in the relative frequency of immunosuppressive regulatory T cells in the aorta (rapa: 70.6±3.0% vs. ctrl: 57.1±3.8%, p<0.05), indicating skewing towards anti-atherogenic immunity. Within the reduced effector T cell pool of rapamycin-treated mice, we observed increased percentages of IL-10 and IFN-γ-expressing CD4+ and CD8+ T cells. In addition, rapamycin diminished the percentage of pro-atherogenic age-associated B cells (rapa: 7.5±1.8% vs. ctrl: 9.2±1.5%, p<0.05) in the mediastinal lymph nodes, and the percentage of pro-atherogenic age-associated B cells in the mediastinal lymph nodes, and reduced the expression of the senescent markers p19 and BTG2 in the spleen. In line with attenuated inflammation we observed a 36% reduction in relative plaque macrophage content in rapamycin-treated mice (rapa: 14.0±3.6% vs. ctrl: 21.8±5.5%, p<0.05). Conclusions Collectively, our study demonstrates that rapamycin treatment in aged atherosclerotic mice promotes regulatory T cells and reduces systemic and plaque accumulation of T cells, ABCs and macrophages. This provides a new understanding of rapamycin in regulating age-associated atherogenic immunity, indicating rapamycin could be a potential therapeutic to halt CVD.

Aging promotes mast cell activation and antigen presenting capacity in atherosclerosis

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Dutch Heart Foundation Aging is an independent and dominant risk factor for atherosclerosis and is associated with a low-grade chronic inflammation termed inflammaging. This age-induced pro-inflammatory environment may have great impact on plaque-residing immune cells, including mast cells. Mast cells have been found to accumulate in the human atherosclerotic plaque upon disease progression and have been associated with plaque instability. However, it is currently unknown whether aging drives the pro-atherogenic effects of mast cells in atherosclerosis. To assess the effects of aging on mast cell phenotype, we examined mast cell populations in young and old male Ldlr-/- mice fed either a chow (CD) or Western-type (WD) diet. We observed a 6-fold increase in the number of mast cells in the aged atherosclerotic aorta compared to young (Y-CD: 4.5±0.7 vs. O-CD: 279.4±9 cells, p<0.01; Y-WD: 45.3±15.2 vs. O-CD: 279.4±9 cells, p<0.01). Furthermore, we detected a marked increase in activation status with age as shown by the significantly augmented number of CD63+ mast cells in the atherosclerotic aorta (Y-CD: 1.8±0.6 vs. O-CD: 259.7±85.4 cells, p<0.01; Y-WD: 41.3±13.6 vs. O-CD: 259.7±85.4 cells, p<0.01). In line, we identified that aged bone-marrow derived mast cells displayed elevated basal CD63 expression compared to young cells, indicative of intrinsic low-grade activation. Moreover, we demonstrated that aging increases the amount of MHCII+ mast cells in atherosclerosis, allowing them to act as antigen-presenting cells, which correspondingly led to increased CD4+ T cell proliferation in vitro (young: 35.2±1.2% vs. old: 45.8±1.2%, p<0.001). Finally, single-cell RNA sequencing of human atherosclerotic plaques confirmed mast cell-specific expression of MHC-II orthologs. To conclude, we established that aging induces phenotypic changes of mast cells in atherosclerosis, regarding both activation status and antigen presenting capacity, which can contribute to disease progression.

Abstract 2142: IL-17 Signaling In Intestinal Epithelial Cells Regulates Microbiota, Metabolism And Neuroinflammation In Atherosclerosis.

Microbiota plays a crucial role in regulating immunity and has been suggested to contribute to atherosclerosis, the precise role of cytokine signaling in intestinal epithelial cells (IEC) in controlling microbiota, inflammation, and vascular disease is poorly understood. Here we investigate the role of IL-17RC signaling in intestinal epithelial cells (IEC) using newly generated Ldlr -/- Il17rc Δ IEC mice. We found that IEC-specific ablation of IL-17RC heightened intestinal inflammation, altered microbiota composition, and led to enhanced atherosclerosis development accompanied by accumulation of activated myeloid and T cells in aortas along with upregulation of genes reminiscent of neuroinflammatory signature. IL-17RC ablation in IEC reduced tight junction proteins contributing to enhanced gut permeability. RNAseq analysis of intestines revealed upregulation of multiple inflammatory pathways, including interferon and TLR signaling, while several metabolic pathways were downregulated in Ldlr -/- Il17rc Δ IEC mice; implying an important role of IL-17R signaling in the control of IEC metabolism, microbes and metabolites at the host-microbiota interface. Metagenomic sequencing revealed alteration of microbiota composition in Ldlr -/- Il17rc Δ IEC . Atherosclerosis development was microbiota dependent as co-housing enhanced the disease in control mice, while microbiota depletion significantly suppressed the disease.Our work uncovers novel protective role of IL-17 signaling in intestine which regulates the barrier, microbiota and metabolism leading to reduced neuroinflammation in atherosclerotic aorta.

Abstract 133: Trem2 Agonist Promotes Atherosclerotic Plaque Stability

Atherosclerosis is an inflammatory disease of the arteries that is a major cause of cardiovascular disease (CVD). Atherosclerotic plaque progression is driven by the deposition of low-density lipoprotein (LDL) within the intima, which is internalized by macrophages, forming lipid-engorged foamy cells. Importantly, the accumulation of excess LDL within foamy macrophages is cytotoxic, driving apoptosis, which ultimately promotes necrotic core formation and increased potential for plaque rupture. Recently, our lab identified the lipid receptor Trem2 to be highly expressed by foamy macrophages and regulate cell survival, proliferation, and cholesterol efflux. Thus, we tested whether agonizing Trem2 could drive macrophage survival to promote plaque stability. Using a Trem2 agonist antibody (AL002a, Alector Inc.), we treated Ldlr -/- mice that had preexisting atherosclerotic plaque with either isotype control or AL002a antibody for 8 weeks. AL002a treated mice showed an increase in plaque size in the aortic arch and aortic sinus. Plaque analysis determined that this was primarily due to an expansion of macrophage cellularity, through promotion of cell survival and proliferation. Furthermore, we found that plaques from AL002a treated mice had decreased necrotic core formation, increased fibrous cap size, and increased collagen deposition, consistent with improved stability. Single cell RNA-sequencing of atherosclerotic aortae from isotype and AL002a treated mice found that Trem2-agonism dramatically reshaped transcriptomes of both foamy macrophages and smooth muscle cells, including upregulation of oxidative phosphorylation (OXPHOS), cholesterol efflux, collagen, and pro-survival genes while downregulating inflammatory pathways. In vitro studies using bone marrow derived macrophages confirmed AL002a’s ability to drive cell survival, proliferation, OXPHOS, and cholesterol efflux in culture. Finally, we determined that AL002a’s ability to reshape aspects of foamy macrophage function was dependent on its ability to activate the kinase SYK downstream of Trem2. Overall, this data identifies Trem2 as a viable therapeutic target for promoting atherosclerotic plaque stability and improving outcomes in CVD.

Interleukin-1 receptor accessory protein blockade limits the development of atherosclerosis and reduces plaque inflammation.

The interleukin-1 receptor accessory protein (IL1RAP) is a co-receptor required for signalling through the IL-1, IL-33, and IL-36 receptors. Using a novel anti-IL1RAP-blocking antibody, we investigated the role of IL1RAP in atherosclerosis. Single-cell RNA sequencing data from human atherosclerotic plaques revealed the expression of IL1RAP and several IL1RAP-related cytokines and receptors, including IL1B and IL33. Histological analysis showed the presence of IL1RAP in both the plaque and adventitia, and flow cytometry of murine atherosclerotic aortas revealed IL1RAP expression on plaque leucocytes, including neutrophils and macrophages. High-cholesterol diet fed apolipoprotein E-deficient (Apoe-/-) mice were treated with a novel non-depleting IL1RAP-blocking antibody or isotype control for the last 6 weeks of diet. IL1RAP blockade in mice resulted in a 20% reduction in subvalvular plaque size and limited the accumulation of neutrophils and monocytes/macrophages in plaques and of T cells in adventitia, compared with control mice. Indicative of reduced plaque inflammation, the expression of several genes related to leucocyte recruitment, including Cxcl1 and Cxcl2, was reduced in brachiocephalic arteries of anti-IL1RAP-treated mice, and the expression of these chemokines in human plaques was mainly restricted to CD68+ myeloid cells. Furthermore, in vitro studies demonstrated that IL-1, IL-33, and IL-36 induced CXCL1 release from both macrophages and fibroblasts, which could be mitigated by IL1RAP blockade. Limiting IL1RAP-dependent cytokine signalling pathways in atherosclerotic mice reduces plaque burden and plaque inflammation, potentially by limiting plaque chemokine production.

Gsα Regulates Macrophage Foam Cell Formation During Atherosclerosis.

Many cardiovascular pathologies are induced by signaling through G-protein-coupled receptors via Gsα (G protein stimulatory α subunit) proteins. However, the specific cellular mechanisms that are driven by Gsα and contribute to the development of atherosclerosis remain unclear. High-throughput screening involving data from single-cell and bulk sequencing were used to explore the expression of Gsα in atherosclerosis. The differentially expression and activity of Gsα were analyzed by immunofluorescence and cAMP measurements. Macrophage-specific Gsα knockout (Mac-GsαKO) mice were generated to study the effect on atherosclerosis. The role of Gsα was determined by transplanting bone marrow and performing assays for foam cell formation, Dil-ox-LDL (oxidized low-density lipoprotein) uptake, chromatin immunoprecipitation, and luciferase reporter assays. ScRNA-seq showed elevated Gnas in atherosclerotic mouse aorta's cholesterol metabolism macrophage cluster, while bulk sequencing confirmed increased GNAS expression in human plaque macrophage content. A significant upregulation of Gsα and active Gsα occurred in macrophages from human and mouse plaques. Ox-LDL could translocate Gsα from macrophage lipid rafts in short-term and promote Gnas transcription through ERK1/2 activation and C/EBPβ phosphorylation via oxidative stress in long-term. Atherosclerotic lesions from Mac-GsαKO mice displayed decreased lipid deposition compared with those from control mice. Additionally, Gsα deficiency alleviated lipid uptake and foam cell formation. Mechanistically, Gsα increased the levels of cAMP and transcriptional activity of the cAMP response element binding protein, which resulted in increased expression of CD36 and SR-A1. In the translational experiments, inhibiting Gsα activation with suramin or cpGN13 reduced lipid uptake, foam cell formation, and the progression of atherosclerotic plaques in mice in vivo. Gsα activation is enhanced during atherosclerotic progression and increases lipid uptake and foam cell formation. The genetic or chemical inactivation of Gsα inhibit the development of atherosclerosis in mice, suggesting that drugs targeting Gsα may be useful in the treatment of atherosclerosis.

Oxidized low-density lipoproteins impair the pro-atherosclerotic effect of granulocyte-macrophage-colony-stimulating factor-producing T helper cells on macrophages.

T cells contribute to the pathogenesis of atherosclerosis. However, the presence and function of granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (ThGM) cells in atherosclerosis development is unknown. This study aims to characterize the phenotype and function of ThGM cells in experimental atherosclerosis. Atherosclerosis was induced by feeding apolipoprotein E knockout (ApoE-/-) mice with a high-fat diet. Aortic ThGM cells were detected and sorted by flow cytometry. The effect of oxidized low-density lipoprotein (oxLDL) on ThGM cells and the impact of ThGM cells on macrophages were evaluated by flow cytometry, quantitative RT-PCR, oxLDL binding/uptake assay, immunoblotting and foam cell formation assay. We found that GM-CSF+IFN-γ- ThGM cells existed in atherosclerotic aortas. Live ThGM cells were enriched in aortic CD4+CCR6-CCR8-CXCR3-CCR10+ T cells. Aortic ThGM cells triggered the expression of interleukin-1β (IL-1β), tumour necrosis factor (TNF), interleukin-6 (IL-6) and C-C motif chemokine ligand 2 (CCL2) in macrophages. Besides, aortic ThGM cells expressed higher CD69 than other T cells and bound to oxLDL. oxLDL suppressed the cytokine expression in ThGM cells probably via inhibiting the signal transducer and activator of transcription 5 (STAT5) signalling. Furthermore, oxLDL alleviated the effect of ThGM cells on inducing macrophages to produce pro-inflammatory cytokines and generate foam cells. The nuclear receptor subfamily 4 group A (NR4A) members NR4A1 and NR4A2 were involved in the suppressive effect of oxLDL on ThGM cells. Collectively, oxLDL suppressed the supportive effect of ThGM cells on pro-atherosclerotic macrophages.

Itaconate suppresses atherosclerosis by activating a Nrf2-dependent antiinflammatory response in macrophages in mice.

Itaconate has emerged as a critical immunoregulatory metabolite. Here, we examined the therapeutic potential of itaconate in atherosclerosis. We found that both itaconate and the enzyme that synthesizes it, aconitate decarboxylase 1 (Acod1, also known as immune-responsive gene 1 [IRG1]), are upregulated during atherogenesis in mice. Deletion of Acod1 in myeloid cells exacerbated inflammation and atherosclerosis in vivo and resulted in an elevated frequency of a specific subset of M1-polarized proinflammatory macrophages in the atherosclerotic aorta. Importantly, Acod1 levels were inversely correlated with clinical occlusion in atherosclerotic human aorta specimens. Treating mice with the itaconate derivative 4-octyl itaconate attenuated inflammation and atherosclerosis induced by high cholesterol. Mechanistically, we found that the antioxidant transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), was required for itaconate to suppress macrophage activation induced by oxidized lipids in vitro and to decrease atherosclerotic lesion areas in vivo. Overall, our work shows that itaconate suppresses atherogenesis by inducing Nrf2-dependent inhibition of proinflammatory responses in macrophages. Activation of the itaconate pathway may represent an important approach to treat atherosclerosis.

Global deficiency in the inflammatory chemokine receptors 1,2,3 and 5 ameliorates atherosclerosis and selectively modulates aortic myeloid cell phenotype

Abstract Background Atherosclerosis is a chronic inflammatory disease in which leukocytes traffic to the vessel wall, a process in part mediated by the inflammatory chemokine receptors (iCCRs): Ccr1, Ccr2, Ccr3 and Ccr5. ICCRs display significant ligand promiscuity and redundancy, which have hampered the definitive deduction of the isolated and collective roles of these receptors. Purpose To assess iCCR expression in human and mouse atherosclerosis and determine the contribution of global iCCR expression to atherosclerosis formation in mice. Methods We derived data from the human carotid endarterectomy biobanks, Athero-Express and Biobank of Karolinska Endarterectomy (BiKE) to investigate iCCR expression. Next, we employed fluorescent reporter mice (iREP) to track iCCR expression via flow cytometry and confocal microscopy. To ascertain the contribution of iCCRs to atherosclerosis, we utilised mice in which the complete iCcr locus incorporating Ccr1, Ccr2, Ccr3 and Ccr5 has been deleted (TACKO mice). We induced atherosclerosis using tail vein injection of AAV8- proprotein convertase subtilisin/kexin type 9 (PCSK9) in combination with 12- or 20-weeks Western diet (WD). Plaque size and composition was assessed via histology while cytometry of time-of-flight (CyTOF) and single-cell RNA-sequencing (scRNA-seq) were used to assess cellular changes. Results By analysing scRNA-seq data from Athero-Express, CCR1 emerged as the most strongly expressed iCCR and was highly localised to macrophages. Next, by utilizing BiKE data, we discovered that CCR1, 2 and 5 expression increased in plaques compared to healthy vessels, with CCR1 and CCR5 more strongly associated with symptomatic patients compared with asymptomatic patients. In iREP mice, CCR1, 2 and 5+ cell numbers were significantly increased in the atherosclerotic aorta compared with healthy mice. Cellular expression levels were variable with CCR2 being dominant in the plaque and increasing markedly on macrophages. Following 12 weeks WD, there was a reduction in mean plaque area within the aortic sinus of TACKO mice (WT 0.20 mm² vs. TACKO 0.10 mm², n=19-20, P<0.0001). Following 20 weeks WD, mean plaque area was also reduced in TACKO mice (WT 0.41 mm² vs. TACKO 0.25 mm², n=9-12, P<0.01) and this was associated with a significant reduction in necrotic core area and a reduction in calcification, reflecting a shift towards a more stable plaque phenotype, as classified according to the Virmani grading system. CyTOF analysis revealed reductions in inflammatory monocytes and conventional type 2 dendritic cells in TACKO aorta accompanied by a phenotypic switch in macrophages towards a less inflammatory phenotype. Finally, scRNA-seq analysis revealed phenotypic differences in non-immune vascular cells between WT and TACKO mice. Conclusion Our results support an athero-protective, plaque-stabilizing effect of iCcr deletion with a selective reduction in inflammatory myeloid cell aortic homing.

Abstract 18579: Endocytic Adaptor Protein DAB2 is a Novel Atheroprotective Regulator in Atherosclerosis

Introduction: Endothelial dysfunction and arterial inflammation are critically important for cardiovascular diseases such as atherosclerosis. Of major importance, they are among the predominant culprits that promote the transition from stable to vulnerable atheromas. Our pilot studies revealed Disabled Homolog 2 (Dab2) deficiency in endothelial cells (EC) results in endothelial dysfunction. However, the molecular mechanisms that direct Dab2 to combat endothelial dysfunction during atherosclerosis are completely unknown. Hypothesis: Dab2 prevents the progress of atherosclerosis by maintaining EC function and reducing inflammatory factors. Methods and Results: We examined the expression of endothelial Dab2 in human atherosclerosis patients and ApoE -/- mice fed a western diet (WD) for 12 weeks by immunoflurecent co-staining of VE-cadherin and Dab2 and observed drastically reduced Dab2 expression in the endothelium and intima of human fatty streaks and mouse atherosclerotic aortae. Genetic deletion of Dab2 in the endothelium of ApoE -/- mice on a WD for 12 weeks significantly increased atherosclerosis as revealed by Oil Red O staining of aortic roots and arches, suggesting that Dab2 could be a atheroprotective flow-responsive gene. Similar results were found in WT and EC-iDab2KO mice injected with AAv8-PCSK9 virus. RNA-seq of primary mouse aortic ECs (MAECs) isolated from WT and EC-Dab2iKO mice revealed Dab2 deficiency upregulated many inflammatory genes. qRT-PCR confirmed TNF-α and IL-6 were significantly upregulated in Dab2KO MAECs. Additionally, we observed an increase in Dab2 binding to NEMO in the absence of endocytic adapter proteins epsins via co-IP with anti-Dab2 in WT and epsin-deficient DKO MAECs. We also found three Klf4 binding sites present in the Dab2 gene promoter/enhancer and that overexpression of Klf4 upregulated Dab2 expression in a dosage-dependent manner. Conclusions: Our findings indicated the essential role of Dab2 in protecting the atherogenic endothelium by reducing macrophage accumulation and increasing plaque stability in the aortic roots, which may provide new anti-atherosclerotic therapeutics.

Single-cell transcriptomics reveals subtype-specific molecular profiles in Nrf2-deficient macrophages from murine atherosclerotic aortas.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional regulator of antioxidant and anti-inflammatory response in all cell types. It also activates the transcription of genes important for macrophage function. Nrf2 activity declines with age and has been closely linked to atherosclerosis, but its specific role in this vascular pathology is not clear. Atherosclerotic plaques contain several macrophage subsets with distinct, yet not completely understood, functions in the lesion development. The aim of this study was to analyze the transcriptome of diverse Nrf2-deficient macrophage subpopulations from murine atherosclerotic aortas. Mice with transcriptionally inactive Nrf2 in Cdh5-expressing cells (Nrf2 Cdh5tKO) were used in the experiments. These mice lack transcriptional Nrf2 activity in endothelial cells, but also in a proportion of leukocytes. We confirmed that the bone marrow-derived and tissue-resident macrophages isolated from Nrf2 Cdh5tKO mice exhibit a significant decline in Nrf2 activity. Atherosclerosis was induced in Nrf2 Cdh5tKO and appropriate control mice via adeno-associated viral vector (AAV)-mediated overexpression of murine proprotein convertase subtilisin/kexin type 9 (Pcsk9) in the liver and high-fat diet feeding. After 21 weeks, live aortic cells were sorted on FACS and single-cell RNA sequencing (scRNA-seq) was performed. Unsupervised clustering singled out 13 distinct aortic cell types. Among macrophages, 9 subclusters were identified. Differential gene expression analysis revealed cell subtype-specific expression patterns. A subset of inflammatory macrophages from atherosclerotic Nrf2 Cdh5tKO mice demonstrated downregulation of DNA replication genes (e.g. Mcm7, Lig1, Pola1) concomitant with upregulation of DNA damage sensor Atr gene. Atherosclerotic Nrf2 Cdh5tKO Lyve1+ resident macrophages showed strong upregulation of IFN-stimulated genes, as well as changes in the expression of death pathways-associated genes (Slc40a1, Bcl2a1). Furthermore, we observed subtype-specific expression of core ferroptosis genes (e.g. Cp, Hells, Slc40a1) in inflammatory versus tissue resident macrophages. This observation suggested a link between ferroptosis and inflammatory microenvironment appearing at a very early stage of atherogenesis. Our findings indicate that Nrf2 deficiency in aortic macrophages leads to subtype-specific transcriptomic changes associated with inflammation, iron homeostasis, cell injury or death pathways. This may help understanding the role of aging-associated decline of Nrf2 activity and the function of specific macrophage subtypes in atherosclerotic lesion development.