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Aims: 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., Methods and Results: 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., Conclusions: 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., Competing Interests: Conflict of interest: M.O. and K.L. are named as co-inventors on pending patents held by the La Jolla Institute for Immunology relating to cardiovascular diagnostics and therapeutics and might have the right to receive royalty payments for inventions or discoveries related to cardiovascular diagnostics or therapeutics. None of the other authors have any competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Aims: Circular RNAs (circRNAs) are considered important regulators of biological processes, but their impact on atherosclerosis development, a key factor in coronary artery disease (CAD), has not been fully elucidated. We aimed to investigate their potential use in patients with CAD and the pathogenesis of atherosclerosis., Methods and Results: Patients with stable angina (SA) or acute coronary syndrome (ACS) and controls were selected for transcriptomic screening and quantification of circRNAs in blood cells. We stained carotid plaque samples for circRNAs and performed gain- and loss-of-function studies in vitro. Western blots, protein interaction analysis, and molecular approaches were used to perform the mechanistic study. ApoE-/- mouse models were employed in functional studies with adeno-associated virus-mediated genetic intervention. We demonstrated elevated circARCN1 expression in peripheral blood mononuclear cells from patients with SA or ACS, especially in those with ACS. Furthermore, higher circARCN1 levels were associated with a higher risk of developing SA and ACS. We also observed elevated expression of circARCN1 in carotid artery plaques. Further analysis indicated that circARCN1 was mainly expressed in monocytes and macrophages, which was also confirmed in atherosclerotic plaques. Our in vitro studies provided evidence that circARCN1 affected the interaction between HuR and ubiquitin-specific peptidase 31 (USP31) mRNA, resulting in attenuated USP31-mediated NF-κB activation. Interestingly, macrophage accumulation and inflammation in atherosclerotic plaques were markedly decreased when circARCN1 was knocked down with adeno-associated virus in macrophages of ApoE-/- mice, while circARCN1 overexpression in the model exacerbated atherosclerotic lesions., Conclusions: Our findings provide solid evidence macrophagic-expressed circARCN1 plays a role in atherosclerosis development by regulating HuR-mediated USP31 mRNA stability and NF-κB activation, suggesting that circARCN1 may serve as a factor for atherosclerotic lesion formation., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Atherosclerosis is a progressive inflammatory disease in which macrophage foam cells play a central role in disease pathogenesis. SPA (surfactant protein A) is a lipid-associating protein involved with regulating macrophage function in various inflammatory diseases. However, the role of SPA in atherosclerosis and macrophage foam cell formation has not been investigated., Methods: SPA expression was assessed in healthy and atherosclerotic human coronary arteries and the brachiocephalic arteries of wild-type or ApoE-deficient mice fed high-fat diets for 4 weeks. Hypercholesteremic wild-type and SPA-deficient mice fed a high-fat diet for 6 weeks were investigated for atherosclerotic lesions in vivo. In vitro experiments using RAW264.7 macrophages, primary resident peritoneal macrophages extracted from wild-type or SPA-deficient mice, and human monocyte-derived macrophages from the peripheral blood of healthy donors determined the functional effects of SPA in macrophage foam cell formation., Results: SPA expression was increased in atherosclerotic lesions in humans and ApoE-deficient mice and in response to a proatherosclerotic stimulus in vitro. SPA deficiency reduced the lipid profiles induced by hypercholesterolemia, attenuated atherosclerosis, and reduced the number of lesion-associated macrophage foam cells. In vitro studies revealed that SPA deficiency reduced intracellular cholesterol accumulation and macrophage foam cell formation. Mechanistically, SPA deficiency dramatically downregulated the expression of scavenger receptor CD36 (cluster of differentiation antigen 36) cellular and lesional expression. Importantly, SPA also increased CD36 expression in human monocyte-derived macrophages., Conclusions: Our results elucidate that SPA is a novel factor promoting atherosclerosis development. SPA enhances macrophage foam cell formation and atherosclerosis by increasing scavenger receptor CD36 expression, leading to increasing cellular OxLDL influx., Competing Interests: None.

Urolithin A (UroA), a dietary phytochemical, is produced by gut bacteria from fruits rich in natural polyphenols ellagitannins (ETs). The efficiency of ETs metabolism to UroA in humans depends on gut microbiota. UroA has shown a variety of pharmacological activities. In this study we investigated the effects of UroA on atherosclerotic lesion development and stability. Apolipoprotein E-deficient (ApoE -/- ) mice were fed a high-fat and high-cholesterol diet for 3 months to establish atherosclerosis model. Meanwhile the mice were administered UroA (50 mg·kg -1 ·d -1 , i.g.). We showed that UroA administration significantly decreased diet-induced atherosclerotic lesions in brachiocephalic arteries, macrophage content in plaques, expression of endothelial adhesion molecules, intraplaque hemorrhage and size of necrotic core, while increased the expression of smooth muscle actin and the thickness of fibrous cap, implying features of plaque stabilization. The underlying mechanisms were elucidated using TNF-α-stimulated human endothelial cells. Pretreatment with UroA (10, 25, 50 μM) dose-dependently inhibited TNF-α-induced endothelial cell activation and monocyte adhesion. However, the anti-inflammatory effects of UroA in TNF-α-stimulated human umbilical vein endothelial cells (HUVECs) were independent of NF-κB p65 pathway. We conducted RNA-sequencing profiling analysis to identify the differential expression of genes (DEGs) associated with vascular function, inflammatory responses, cell adhesion and thrombosis in UroA-pretreated HUVECs. Human disease enrichment analysis revealed that the DEGs were significantly correlated with cardiovascular diseases. We demonstrated that UroA pretreatment mitigated endothelial inflammation by promoting NO production and decreasing YAP/TAZ protein expression and TEAD transcriptional activity in TNF-α-stimulated HUVECs. On the other hand, we found that UroA administration modulated the transcription and cleavage of lipogenic transcription factors SREBP1/2 in the liver to ameliorate cholesterol metabolism in ApoE -/- mice. This study provides an experimental basis for new dietary therapeutic option to prevent atherosclerosis., (© 2024. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

Background: Atherosclerotic (AS) plaques require a dense necrotic core and a robust fibrous cap to maintain stability. While previous studies have indicated that the traditional Chinese medicine Huang Lian Jie Du Decoction (HLJDD) possesses the capability to stabilize AS plaques, the underlying mechanisms remain obscure. This study aims to delve deeper into the potential mechanisms by which HLJDD improves AS through an integrated research strategy., Methods: Leveraging an AS model in ApoE -/- mice exposed to a high-fat diet (HFD), we scrutinized the therapeutic effects of HLJDD using microscopic observations, oil red O staining, HE staining and Masson staining. Employing comprehensive techniques of network pharmacology, bioinformatics, and molecular docking, we elucidated the mechanism by which HLJDD stabilizes AS plaques. In vitro experiments, utilizing ox-LDL-induced macrophages and apoptotic vascular smooth muscle cells (VSMCs), assessed the impact of HLJDD on efferocytosis and the role of SLC2A1., Results: In vivo experiments showcased the efficacy of HLJDD in reducing the quantity of aortic plaques, diminishing lipid deposition, and enhancing plaque stability in AS mice. Employing network pharmacology and machine learning, we pinpointed SLC2A1 as a crucial regulatory target. Molecular docking further validated the binding of HLJDD components with SLC2A1. The experiments demonstrated a dose-dependent upregulation in SLC2A1 expression by HLJDD, amplifying efferocytosis. Importantly, this effect was reversed by the SLC2A1 inhibitor STF-31, highlighting the pivotal role of SLC2A1 as a target., Conclusion: The HLJDD can modulate macrophage efferocytosis by enhancing the expression levels of SLC2A1, thereby improving the stability of atherosclerotic plaques., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Aims: 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., Methods and Results: 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., Conclusion: 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., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background and Aims: CCN4/WISP-1 regulates various cell behaviours that contribute to atherosclerosis progression, including cell adhesion, migration, proliferation and survival. We therefore hypothesised that CCN4 regulates the development and progression of atherosclerotic plaques., Methods: We used a high fat fed ApoE -/- mouse model to study atherosclerotic plaque progression in the brachiocephalic artery and aortic root. In protocol 1, male ApoE -/- mice with established plaques were given a CCN4 helper-dependent adenovirus to see the effect of treatment with CCN4, while in protocol 2 male CCN4 -/- ApoE -/- were compared to CCN4 +/+ ApoE -/- mice to assess the effect of CCN4 deletion on plaque progression., Results: CCN4 overexpression resulted in reduced occlusion of the brachiocephalic artery with less apoptosis, fewer macrophages, and attenuated lipid core size. The amount of plaque found on the aortic root was also reduced. CCN4 deficiency resulted in increased apoptosis and occlusion of the brachiocephalic artery as well as increased plaque in the aortic root. Additionally, in vitro cells from CCN4 -/- ApoE -/- mice had higher apoptotic levels. CCN4 deficiency did not significantly affect blood cholesterol levels or circulating myeloid cell populations., Conclusions: We conclude that in an atherosclerosis model the most important action of CCN4 is the effect on cell apoptosis. CCN4 provides pro-survival signals and leads to reduced cell death, lower macrophage number, smaller lipid core size and reduced atherosclerotic plaque burden. As such, the pro-survival effect of CCN4 is worthy of further investigation, in a bid to find a therapeutic for atherosclerosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Aims: Mast cell-derived heparin proteoglycans (HEP-PG) can be mimicked by bioconjugates carrying antithrombotic and anti-inflammatory properties. The dual antiplatelet and anticoagulant (APAC) construct administered, either locally or intravenously (i.v.), targets activated endothelium, its adhesion molecules, and subendothelial matrix proteins, all relevant to atherogenesis. We hypothesized that APAC influences cellular interactions in atherosclerotic lesion development and studied APAC treatment during the initiation and progression of experimental atherosclerosis., Methods: Male western-type diet-fed Apoe -/- mice were equipped with perivascular carotid artery collars to induce local atherosclerosis. In this model, mRNA expression of adhesion molecules including ICAM-1, VCAM-1, P-Selectin, and Platelet Factor 4 (PF4) are upregulated upon lesion development. From day 1 (prevention) or from 2.5 weeks after lesion initiation (treatment), mice were administered 0.2 mg/kg APAC i.v. or control vehicle three times weekly for 2.5 weeks. At week 5 after collar placement, mice were sacrificed, and lesion morphology was microscopically assessed., Results: APAC treatment did not affect body weight or plasma total cholesterol levels during the experiments. In the prevention setting, APAC reduced carotid artery plaque size and volume by over 50 %, aligning with decreased plaque macrophage area and collagen content. During the treatment setting, APAC reduced macrophage accumulation and necrotic core content, and improved markers of plaque stability., Conclusions: APAC effectively reduced early atherosclerotic lesion development and improved markers of plaque inflammation in advanced atherosclerosis. Thus, APAC may have potential to alleviate the progression of atherosclerosis., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: RL is the CSO and CMO of Aplagon Ltd. The other authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Aims: Atherosclerotic cardiovascular disease complicated by diabetes mellitus (DM) is the leading cause of death in diabetic patients, and it is strongly associated with macrophages and inflammasomes. It has been found that activation of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome is closely associated with phosphatidylinositol 4-phosphate (PI4P) on the trans-Golgi. However, how PI4P and NLRP3 regulate macrophage function and its role in diabetic atherosclerotic plaques is unclear., Methods: The expression of Pi4p and Nlrp3-inflammasome-related proteins in atherosclerosis in apolipoprotein E-deficient (Apoe -/- ) and Apoe -/- DM mice was investigated. Then, Pi4p levels were affected by shRNA-Pi4kb or cDNA-Sac1 plasmid to investigate the effects of changes in Pi4p-related metabolic enzymes on macrophage function. Finally, genetically modified macrophages were injected into diabetic Apoe -/- mice to explore the effects on atherosclerosis., Results: DM promoted plaque progression in atherosclerotic mice and increased expression of Pi4p and Nlrp3 in plaques. In addition, impaired macrophage function induced by high glucose was reversed by transfected shRNA-Pi4kb or cDNA-Sac1 plasmid. Furthermore, decreased levels of Pi4p reduced plaque area in diabetic Apoe -/- mice., Conclusions: Our data suggests that Pi4p/Nlrp3 in macrophages play an important role in the exacerbation of atherosclerosis in diabetic mice. Pi4p-related metabolizing enzymes (PI4KB and SAC1) may be a potential therapeutic strategy for diabetic atherosclerosis, and macrophage therapy is also a potential treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Clinical evidence suggests anti-Hsp60 antibodies could contribute to atherosclerosis (AS) development, with unclear mechanisms. This study aims to explore the role of anti-HSP60-mediated autoimmunity in AS progression. HSP60-MHC tetramers were used to characterize HSP60-specific CD4 + T cells and assess TCR responses in mice. These cells were transplanted into AS mice to examine immune cell differentiation and infiltration in plaques and blood. Mice were injected with recombinant HSP60 or anti-HSP60 sera to evaluate effects on plaque progression and macrophage activity. Experiments with muMT -/- Apoe -/- mice examined humoral immunity's role in this autoimmunity. HSP60-reactive CD4 + T cells in AS mice differentiated into follicular helper cells, not Th1/Th17. Anti-HSP60 treatments increased macrophage infiltration and M1 polarization, indicating an anti-HSP60-driven inflammatory progression, dependent on humoral immunity. Anti-HSP60 influences macrophage infiltration, polarization, and plaque formation via humoral immunity, shedding light on its potential role in AS progression., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: To explore the feasibility of using cRGD-GNR-PFP-NPs to assess plaque vulnerability in an atherosclerotic plaque mouse model by dual-modal photoacoustic/ultrasonic imaging., Methods: A nanomolecular probe containing gold nanorods (GNRs) and perfluoropentane (PFP) coated with the cyclic Arg-Gly-Asp (cRGD) peptide were prepared by double emulsion solvent evaporation and carbodiimide methods. The morphology, particle size, potential, cRGD conjugation and absorption features of the nanomolecular probe were characterized, along with its in vitro phase transformation and photoacoustic/ultrasonic dual-modal imaging properties. In vivo fluorescence imaging was used to determine the distribution of cRGD-GNR-PFP-NPs in vivo in apolipoprotein E-deficient (ApoE -/- ) atherosclerotic plaque model mice, the optimal imaging time was determined, and photoacoustic/ultrasonic dual-modal molecular imaging of integrin αvβ3 expressed in atherosclerotic plaques was performed. Pathological assessments verified the imaging results in terms of integrin αvβ3 expression and plaque vulnerability., Results: cRGD-GNR-PFP-NPs were spherical with an appropriate particle size (average of approximately 258.03±6.75 nm), a uniform dispersion, and a potential of approximately -9.36±0.53 mV. The probe had a characteristic absorption peak at 780~790 nm, and the surface conjugation of the cRGD peptide reached 92.79%. cRGD-GNR-PFP-NPs were very stable in the non-excited state but very easily underwent phase transformation under low-intensity focused ultrasound (LIFU) and had excellent photoacoustic/ultrasonic dual-modal imaging capability. Mice fed a high-fat diet for 20 weeks had obvious hyperlipidemia with larger, more vulnerable plaques. These plaques could be specifically targeted by cRGD-GNR-PFP-NPs as determined by in vivo fluorescence imaging, and the enrichment of nanomolecular probe increased with the increasing of plaque vulnerability; the photoacoustic/ultrasound signals of the plaques in the high-fat group were stronger. The pathological assessments were in good agreement with the cRGD-GNR-PFP-NPs plaque accumulation, integrin αvβ3 expression and plaque vulnerability results., Conclusion: A phase variant photoacoustic/ultrasonic dual-modal cRGD nanomolecular probe was successfully prepared and can be used to identify plaque vulnerability safely and effectively., Competing Interests: The authors declare that there are no conflicts of interest in this work., (© 2024 Yu et al.)

Background: Visceral adipose tissue in individuals with obesity is an independent cardiovascular risk indicator. However, it remains unclear whether adipose tissue influences common cardiovascular diseases, such as atherosclerosis, through its secreted exosomes., Methods: The exosomes secreted by adipose tissue from diet-induced obesity mice were isolated to examine their impact on the progression of atherosclerosis and the associated mechanism. Endothelial apoptosis and the proliferation and migration of vascular smooth muscle cells (VSMCs) within the atherosclerotic plaque were evaluated. Statistical significance was analyzed using GraphPad Prism 9.0 with appropriate statistical tests., Results: We demonstrate that adipose tissue-derived exosomes (AT-EX) exacerbate atherosclerosis progression by promoting endothelial apoptosis, proliferation, and migration of VSMCs within the plaque in vivo. MicroRNA-132/212 (miR-132/212) was detected within AT-EX cargo. Mechanistically, miR-132/212-enriched AT-EX exacerbates palmitate acid-induced endothelial apoptosis via targeting G protein subunit alpha 12 and enhances platelet-derived growth factor type BB-induced VSMC proliferation and migration by targeting phosphatase and tensin homolog in vitro. Importantly, melatonin decreases exosomal miR-132/212 levels, thereby mitigating the pro-atherosclerotic impact of AT-EX., Conclusion: These data uncover the pathological mechanism by which adipose tissue-derived exosomes regulate the progression of atherosclerosis and identify miR-132/212 as potential diagnostic and therapeutic targets for atherosclerosis., (© 2024. The Author(s).)

Background and Aims: In advanced atherosclerotic lesions, macrophage deaths result in necrotic core formation and plaque vulnerability. Cyclophilin D (CypD) is a mitochondria-specific cyclophilin involved in the process of cell death after organ ischemia-reperfusion. However, the role of CypD in atherosclerosis, especially in necrotic core formation, is unknown. Therefore, this experiment aims to clarify the role of CypD in necrotic core formation., Methods: To clarify the specific role of CypD, encoded by Ppif in mice, apolipoprotein-E/CypD-double knockout (Apoe -/- Ppif -/- ) mice were generated. These mice were fed a high-fat diet containing 0.15 % cholesterol for 24 weeks to accelerate atherosclerotic lesion development., Results: Deletion of CypD decreased the necrotic core size, accompanied by a reduction of macrophage apoptosis compared to control Apoe -/- mice. In RAW264.7 cells, siRNA-mediated knockdown of CypD attenuated the release of cytochrome c from the mitochondria to the cytosol induced by endoplasmic reticulum stress inducer thapsigargin. In addition, necroptosis, induced by TNF-α and caspase inhibitor, was attenuated by knockdown of CypD. Ly-6C high inflammatory monocytes in peripheral blood leukocytes and mRNA expression of Il1b in the aorta were decreased by deletion of CypD. In contrast, siRNA-mediated knockdown of CypD did not significantly decrease Il1b nor Ccl2 mRNA expression in RAW264.7 cells treated with LPS and IFN-γ, suggesting that inhibition of inflammation in vivo is likely due to decreased cell death in the atherosclerotic lesions rather than a direct action of CypD deletion on the macrophage., Conclusions: These results indicate that CypD induces macrophage death and mediates necrotic core formation in advanced atherosclerotic lesions. CypD could be a novel therapeutic target for treating atherosclerotic vascular diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

There is a large body of evidence that cellular metabolism governs inflammation, and that inflammation contributes to the progression of atherosclerosis. However, whether mitochondrial DNA synthesis affects macrophage function and atherosclerosis pathology is not fully understood. Here we show, by transcriptomic analyzes of plaque macrophages, spatial single cell transcriptomics of atherosclerotic plaques, and functional experiments, that mitochondrial DNA (mtDNA) synthesis in atherosclerotic plaque macrophages are triggered by vascular cell adhesion molecule 1 (VCAM-1) under inflammatory conditions in both humans and mice. Mechanistically, VCAM-1 activates C/EBPα, which binds to the promoters of key mitochondrial biogenesis genes - Cmpk2 and Pgc1a. Increased CMPK2 and PGC-1α expression triggers mtDNA synthesis, which activates STING-mediated inflammation. Consistently, atherosclerosis and inflammation are less severe in Apoe -/- mice lacking Vcam1 in macrophages. Downregulation of macrophage-specific VCAM-1 in vivo leads to decreased expression of LYZ1 and FCOR, involved in STING signalling. Finally, VCAM-1 expression in human carotid plaque macrophages correlates with necrotic core area, mitochondrial volume, and oxidative damage to DNA. Collectively, our study highlights the importance of macrophage VCAM-1 in inflammation and atherogenesis pathology and proposes a self-acerbating pathway involving increased mtDNA synthesis., (© 2024. The Author(s).)

The effects of calcitonin gene-related peptide (CGRP) on atherosclerosis remain unclear. We used apolipoprotein E-deficient (ApoE -/- ) mice to generate double-knockout ApoE -/- :CGRP -/- mice lacking alpha CGRP. ApoE -/- :CGRP -/- mice exhibited larger atherosclerotic plaque areas, peritoneal macrophages with enhanced migration functions, and elevated levels of the inflammatory cytokine tumor necrosis factor (TNF)-⍺. Thus, we also explored whether inhibiting TNF-⍺ could improve atherosclerosis in ApoE -/- :CGRP -/- mice by administering etanercept intraperitoneally once a week (5 mg/kg) alongside a high-fat diet for 2 weeks. This treatment led to significant reductions in aortic root lesion size, atherosclerotic plaque area and macrophage migration in ApoE -/- :CGRP -/- mice compared with mice treated with human IgG (5 mg/kg). We further examined whether results observed in ApoE -/- :CGRP -/- mice could similarly be obtained by administering a humanized monoclonal CGRP antibody, galcanezumab, to ApoE -/- mice. ApoE -/- mice were subcutaneously administered galcanezumab at an initial dose of 50 mg/kg, followed by a dose of 30 mg/kg in the second week. Galcanezumab administration did not affect systolic blood pressure, serum lipid levels, or macrophage migration but led to a significant increase in lipid deposition at the aortic root. These findings suggest that alpha CGRP plays a critical role in inhibiting the progression of atherosclerosis., (© 2024. The Author(s).)

Vulnerable atherosclerotic plaque rupture, the leading cause of fatal atherothrombotic events, is associated with an increased risk of mortality worldwide. Peroxisome proliferator-activated receptor delta (PPARδ) has been shown to modulate vascular smooth muscle cell (SMC) phenotypic switching, and, hence, atherosclerotic plaque stability. Melatonin reportedly plays a beneficial role in cardiovascular diseases; however, the mechanisms underlying improvements in atherosclerotic plaque vulnerability remain unknown. In this study, we assessed the role of melatonin in regulating SMC phenotypic switching and its consequential contribution to the amelioration of atherosclerotic plaque vulnerability and explored the mechanisms underlying this process. We analyzed features of atherosclerotic plaque vulnerability and markers of SMC phenotypic transition in high-cholesterol diet (HCD)-fed apolipoprotein E knockout (ApoE -/- ) mice and human aortic SMCs (HASMCs). Melatonin reduced atherosclerotic plaque size and necrotic core area while enhancing collagen content, fibrous cap thickness, and smooth muscle alpha-actin positive cell coverage on the plaque cap, which are all known phenotypic characteristics of vulnerable plaques. In atherosclerotic lesions, melatonin significantly decreased the synthetic SMC phenotype and KLF4 expression and increased the expression of PPARδ, but not PPARα and PPARγ, in HCD-fed ApoE -/- mice. These results were subsequently confirmed in the melatonin-treated HASMCs. Further analysis using PPARδ silencing and immunoprecipitation assays revealed that PPARδ plays a role in the melatonin-induced SMC phenotype switching from synthetic to contractile. Collectively, we provided the first evidence that melatonin mediates its protective effect against plaque destabilization by enhancing PPARδ-mediated SMC phenotypic switching, thereby indicating the potential of melatonin in treating atherosclerosis., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In this study, we investigated the protective role of Mzb1 in atherosclerotic plaque vulnerability. To explore the impact of Mzb1, we analyzed Mzb1 expression, assessed apoptosis, and evaluated mitochondrial function in atherosclerosis (AS) mouse models and human vascular smooth muscle cells (HVSMCs). We observed a significant decrease in Mzb1 expression in AS mouse models and ox-LDL-treated HVSMCs. Downregulation of Mzb1 increased ox-LDL-induced apoptosis and cholesterol levels of HVSMCs, while Mzb1 overexpression alleviated these effect. Mzb1 was found to enhance mitochondrial function, as evidenced by restored ATP synthesis, mitochondrial membrane potential, and reduced mtROS production. Moreover, Mzb1 overexpression attenuated atherosclerotic plaque vulnerability in ApoE -/- mice. Our findings suggest that Mzb1 overexpression regulates the AMPK/SIRT1 signaling pathway, leading to the attenuation of atherosclerotic plaque vulnerability. This study provides compelling evidence for the protective effect of Mzb1 on atherosclerotic plaques by alleviating apoptosis and modulating mitochondrial function in ApoE -/- mice., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Cell phenotype switching is increasingly being recognized in atherosclerosis. However, our understanding of the exact stimuli for such cellular transformations and their significance for human atherosclerosis is still evolving. Intraplaque hemorrhage is thought to be a major contributor to plaque progression in part by stimulating the influx of CD163 + macrophages. Here, we explored the hypothesis that CD163 + macrophages cause plaque progression through the induction of proapoptotic endothelial-to-mesenchymal transition (EndMT) within the fibrous cap., Methods: Human coronary artery sections from CVPath's autopsy registry were selected for pathological analysis. Athero-prone ApoE -/- and ApoE -/- /CD163 -/- mice were used for in vivo studies. Human peripheral blood mononuclear cell-induced macrophages and human aortic endothelial cells were used for in vitro experiments., Results: In 107 lesions with acute coronary plaque rupture, 55% had pathological evidence of intraplaque hemorrhage in nonculprit vessels/lesions. Thinner fibrous cap, greater CD163 + macrophage accumulation, and a larger number of CD31/FSP-1 (fibroblast specific protein-1) double-positive cells and TUNEL (terminal deoxynucleotidyl transferase-dUTP nick end labeling) positive cells in the fibrous cap were observed in nonculprit intraplaque hemorrhage lesions, as well as in culprit rupture sections versus nonculprit fibroatheroma sections. Human aortic endothelial cells cultured with supernatants from hemoglobin/haptoglobin-exposed macrophages showed that increased mesenchymal marker proteins (transgelin and FSP-1) while endothelial markers (VE-cadherin and CD31) were reduced, suggesting EndMT induction. Activation of NF-κB (nuclear factor kappa β) signaling by proinflammatory cytokines released from CD163 + macrophages directly regulated the expression of Snail, a critical transcription factor during EndMT induction. Western blot analysis for cleaved caspase-3 and microarray analysis of human aortic endothelial cells indicated that apoptosis was stimulated during CD163 + macrophage-induced EndMT. Additionally, CD163 deletion in athero-prone mice suggested that CD163 is required for EndMT and plaque progression. Using single-cell RNA sequencing from human carotid endarterectomy lesions, a population of EndMT was detected, which demonstrated significant upregulation of apoptosis-related genes., Conclusions: CD163 + macrophages provoke EndMT, which may promote plaque progression through fibrous cap thinning., Competing Interests: R. Virmani and A.V. Finn have received institutional research support from R01 HL141425 Leducq Foundation Grant, the National Institutes of Health Grant (NIH; HL141425), the Amazon Web Services Coronavirus Disease 2019 Diagnostic Development Initiative Grant, 480 Biomedical, 4C Medical, 4Tech, Abbott Vascular, Ablative Solutions, Absorption Systems, Advanced NanoTherapies, Aerwave Medical, Alivas, Amgen, Asahi Medical, Aurios Medical, Avantec Vascular, BD, Biosensors, Biotronik, Biotyx Medical, Bolt Medical, Boston Scientific, Canon, Cardiac Implants, Cardiawave, CardioMech, Cardionomic, Celonova, Cerus EndoVascular, Chansu Vascular Technologies, Childrens National, Concept Medical, Cook Medical, Cooper Health, Cormaze, CRL, Croivalve, Cardiovascular Systems, Inc. (CSI), Dexcom, Edwards Lifesciences, Elucid Bioimaging, eLum Technologies, Emboline, Endotronix, Envision, Filterlex, Imperative Care, Innovalve, Innovative, Cardiovascular Solutions, Intact Vascular, Interface Biologics, Intershunt Technologies, Invatin, Lahav, Limflow, L&J Bio, Lutonix, Lyra Therapeutics, Mayo Clinic, Maywell, MDS, MedAlliance, Medanex, Medtronic, Mercator, Microport, Microvention, Neovasc, Nephronyx, Nova Vascular, Nyra Medical, Occultech, Olympus, Ohio Health, OrbusNeich, Ossiso, Phenox, Pi-Cardia, Polares Medical, Polyvascular, Profusa, ProKidney, LLC, Protembis, Pulse Biosciences, Qool Therapeutics, Recombinetics, Recor Medical, Regencor, Renata Medical, Restore Medical, Ripple Therapeutics, Rush University, Sanofi, Shockwave, SMT, SoundPipe, Spartan Micro, Spectrawave, Surmodics, Terumo Corporation, The Jacobs Institute, Transmural Systems, Transverse Medical, TruLeaf, University of California, San Francisco (UCSF), University of Pittsburgh Medical Center (UPMC), Vascudyne, Vesper, Vetex Medical, Whiteswell, WL Gore, and Xeltis. A.V. Finn received honoraria from Abbott Vascular, Biosensors, Boston Scientific, Celonova, Cook Medical, CSI, Lutonix Bard, Sinomed, and Terumo Corporation and is a consultant for Amgen, Abbott Vascular, Boston Scientific, Celonova, Cook Medical, Lutonix Bard, and Sinomed. R. Virmani received honoraria from Abbott Vascular, Biosensors, Boston Scientific, Celonova, Cook Medical, Cordis, CSI, Lutonix Bard, Medtronic, OrbusNeich Medical, CeloNova, SINO Medical Technology, ReCore, Terumo Corporation, W. L. Gore, and Spectranetics and is a consultant for Celonova, Cook Medical, CSI, Edwards Lifesciences, Bard BD, Medtronic, OrbusNeich Medical, ReCor Medical, SinoMedical Sciences Technology, Surmodics, Terumo Corporation, W. L. Gore, and Xeltis. L. Guo is supported by NIH (grant HL141425) and the Leducq Foundation Grant. M. Mori, A. Sakamoto, and K. Kawai are supported by the Leducq Foundation Grant. The other authors report no conflicts.

Abstract: Atherosclerosis (AS) is a chronic progressive disease caused by various factors and causes various cerebrovascular and cardiovascular diseases (CVDs). Reducing the plasma levels of low-density lipoprotein cholesterol is the primary goal in preventing and treating AS. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a crucial role in regulating low-density lipoprotein cholesterol metabolism. Panax notoginseng has potent lipid-reducing effects and protects against CVDs, and its saponins induce vascular dilatation, inhibit thrombus formation, and are used in treating CVDs. However, the anti-AS effect of the secondary metabolite, 20( S )-protopanaxatriol (20( S )-PPT), remains unclear. In this study, the anti-AS effect and molecular mechanism of 20( S )-PPT were investigated in vivo and in vitro by Western blotting, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence staining, and other assays. The in vitro experiments revealed that 20( S )-PPT reduced the levels of PCSK9 in the supernatant of HepG2 cells, upregulated low-density lipoprotein receptor protein levels, promoted low-density lipoprotein uptake by HepG2 cells, and reduced PCSK9 mRNA transcription by upregulating the levels of forkhead box O3 protein and mRNA and decreasing the levels of HNF1α and SREBP2 protein and mRNA. The in vivo experiments revealed that 20( S )-PPT upregulated aortic α-smooth muscle actin expression, increased the stability of atherosclerotic plaques, and reduced aortic plaque formation induced by a high-cholesterol diet in ApoE -/- mice (high-cholesterol diet-fed group). Additionally, 20( S )-PPT reduced the aortic expression of CD68, reduced inflammation in the aortic root, and alleviated the hepatic lesions in the high-cholesterol diet-fed group. The study revealed that 20( S )-PPT inhibited low-density lipoprotein receptor degradation via PCSK9 to alleviate AS., Competing Interests: The authors report no conflicts of interest., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Ethnopharmacological Relevance: Zexieyin formula (ZXYF), a traditional Chinese herbal formula recorded in the Huangdi Neijing to have efficacy in relieving spleen dampness and heat accumulation syndrome, which is also the key pathogenesis of atherosclerosis (AS). The efficacy has demonstrated by our previous studies. However, the intrinsic mechanism of ZXYF for treating vascular inflammation and the effect of inflammatory response on plaque are not known. Currently, plaque stabilization is crucial for the prognosis of AS., Aim of the Study: Our study mainly focused on the therapeutic effects of ZXYF on high-fat diet (HFD)-induced vascular inflammation and vulnerable plaques (VP) in mice and explored its underlying mechanism., Methods and Materials: Male apolipoprotein E knockout (APOE-/-) mice were fed HFD for 8 weeks to establish a VP model. During this period, the mice were also administered ZXYF, while atorvastatin (ATO) was used as a positive control. Aortic plaque area and morphology were detected by oil red staining and HE staining. Aortic plaque collagen content was detected by Masson staining. M1/M2 type macrophages were detected using immunofluorescence (IF). The study analyzed the levels of inflammation-related cytokines (IL-1β, IL-10, IL-6), MAPK/NF-κB pathway proteins, and NLRP3 inflammasomes (NLRP3, Caspase-1) using Western blot. Additionally, the levels of matrix metalloproteinase (MMP)-2 and MMP-9 and α-smooth muscle actin (α-SMA) in the aorta were analyzed using immunohistochemistry (IHC). The plaque instability index was calculated for each group using the vulnerable plaque formula., Results: In this study, APOE-/- mice were fed high-fat diet for 8 weeks. The results of oil-red and HE staining indicated a significant increase in the aortic plaque area of the mice, which exhibited a typical VP phenotype. ZXYF and ATO significantly improved AS plaques and prevented plaque rupture. HFD exacerbated vascular inflammation, stimulated macrophage conversion to M1-type through the MAPK/NF-κB signaling pathway, and released pro-inflammatory factors such as interleukin (IL)-1β, IL-1α, and IL-6. These factors activated NLRP3 inflammasome, leading to cellular death. However, ZXYF could reverse this trend and promote the conversion of macrophages to the anti-inflammatory M2 type. The anti-inflammatory effect of ATO was not significant. Moreover, HFD promoted the release of MMP-2 and MMP-9 from macrophages, which degraded plaque collagen, and induced a decrease in plaque SMC content, resulting in a thinning of the plaque fibrous cap. In contrast, ZXYF inhibited the decomposition of plaque collagen and increased the content of plaque smooth muscle cells (SMC) by reducing macrophage secretion of MMPs, thereby stabilizing plaques. Although ATO could reverse the decrease in plaque collagen and SMC content, its effect on MMPs was not significant. Finally, we calculated the vulnerability index to assess the overall risk of the plaque vulnerability phenotype. In line with these findings, ZXYF and ATO were able to effectively reverse the increase in the vulnerability index caused by HFD and lower the risk of adverse cardiovascular events., Conclusion: Our results suggested that ZXYF could reduce inflammation and increase plaque stability by inhibiting the MAPK/NF-κB signaling pathway, which provided a theoretical basis for clinical application and subsequent research., Competing Interests: Declaration of competing interest The authors declare there is no commercial or financial relationships that could be construed as a potential conflict of interest regarding this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Thromboembolic events secondary to rupture or erosion of advanced atherosclerotic lesions is the global leading cause of death. The most common and effective means to reduce these major adverse cardiovascular events, including myocardial infarction and stroke, is aggressive lipid lowering via a combination of drugs and dietary modifications. However, we know little regarding the effects of reducing dietary lipids on the composition and stability of advanced atherosclerotic lesions, the mechanisms that regulate these processes, and what therapeutic approaches might augment the benefits of lipid lowering., Methods: Smooth muscle cell lineage-tracing Apoe -/- mice were fed a high-cholesterol Western diet for 18 weeks and then a zero-cholesterol standard laboratory diet for 12 weeks before treating them with an IL (interleukin)-1β or control antibody for 8 weeks. We assessed lesion size and remodeling indices, as well as the cellular composition of aortic and brachiocephalic artery lesions, indices of plaque stability, overall plaque burden, and phenotypic transitions of smooth muscle cell and other lesion cells by smooth muscle cell lineage tracing combined with single-cell RNA sequencing, cytometry by time-of-flight, and immunostaining plus high-resolution confocal microscopic z-stack analysis., Results: Lipid lowering by switching Apoe -/- mice from a Western diet to a standard laboratory diet reduced LDL cholesterol levels by 70% and resulted in multiple beneficial effects including reduced overall aortic plaque burden, as well as reduced intraplaque hemorrhage and necrotic core area. However, contrary to expectations, IL-1β antibody treatment after diet-induced reductions in lipids resulted in multiple detrimental changes including increased plaque burden and brachiocephalic artery lesion size, as well as increasedintraplaque hemorrhage, necrotic core area, and senescence as compared with IgG control antibody-treated mice. Furthermore, IL-1β antibody treatment upregulated neutrophil degranulation pathways but downregulated smooth muscle cell extracellular matrix pathways likely important for the protective fibrous cap., Conclusions: Taken together, IL-1β appears to be required for the maintenance of standard laboratory diet-induced reductions in plaque burden and increases in multiple indices of plaque stability., Competing Interests: Disclosures None.

Objective:  Aldehyde dehydrogenase 4A1 (ALDH4A1) was recently reported to be a novel autoantigen of atherosclerosis. However, its role in different phenotypes of acute coronary syndrome remains unclear. Herein, we planned to explore the circulating and regional expression of ALDH4A1 in patients with plaque rupture (PR) and plaque erosion (PE) determined by optical coherence tomography (OCT)., Methods and Results:  After applying the inclusion and exclusion criteria, a prospective series of 312 patients with ST segment elevated myocardial infarction (STEMI), including 161 patients with PR and 151 patients with PE determined by OCT, were enrolled for plasma ALDH4A1 testing. In addition, ALDH4A1 was quantified using immunofluorescence in aspirated coronary thrombus samples obtained from 31 patients with PR and 25 patients with PE. In addition, we established an atherosclerosis mouse model and analyzed the distribution of ALDH4A1 expression in different mouse organs. Furthermore, we compared the level of ALDH4A1 in the spleen and carotid artery between Apoe -/- and C57 mice. The results showed that the plasma level of ALDH4A1 was significantly higher in STEMI patients with PE than in those with PR (4.6 ng/mL [2.2-8.7] vs. 3.5 ng/mL [1.6-5.6] p  = 0.005). The expression of ALDH4A1 in aspirated coronary thrombi was also significantly higher in patients with PE than in those with PR (mean gray value: 32.0 [23.6-40.6] vs. 16.8 [14.0-24.5], p  < 0.001). In animal models, the expression of ALDH4A1 is much higher in the spleen than in other organs, and the level of ALDH4A1 is significantly elevated in the spleen and carotid artery of Apoe -/- mice compared with C57 mice., Conclusion:  The high levels of ALDH4A1 in the plasma and aspirated coronary thrombi independently correlated with PE in patients with STEMI. These results suggested that ALDH4A1 is involved in the mechanism of PE and serves as a promising biomarker and treatment target for patients with PE., Competing Interests: None declared., (Thieme. All rights reserved.)

Background:  Intra-plaque hemorrhage (IPH) leads to rapid plaque progression and instability through upregulation of matrix metalloproteinases (MMPs) and collagen degradation. Hemoglobin-derived hemin during IPH promotes plaque instability. We investigated whether hemin affects MMP overexpression in macrophages and explored the underlying mechanisms., Material and Methods:  In vivo, hemorrhagic plaque models were established in rabbits and ApoE -/- mice. Ferrostatin-1 was used to inhibit ferroptosis. Plaque size, collagen, and MMP2/9 levels were evaluated using immunohistochemistry, H&E, Sirius Red, and Masson staining. In vitro, mouse peritoneal macrophages were extracted. Western blot and ELISA were used to measure MMP2/9 levels. Bioinformatics analysis investigated the association between MMPs and ferroptosis pathway genes. Macrophage ferroptosis was assessed by evaluating cell viability, lipid reactive oxygen species, mitochondrial ultrastructure, iron content, and COX2 levels after pretreatment with cell death inhibitors. Hemin's impact on ferroptosis and MMP expression was studied using Ferrostatin-1 and SB202190., Results:  In the rabbit hemorrhagic plaques, hemin deposition and overexpression of MMP2/9 were observed, particularly in macrophage-enriched regions. In vitro, hemin induced ferroptosis and MMP2/9 expression in macrophages. Ferrostatin-1 and SB202190 inhibited hemin-induced MMP2/9 overexpression. Ferrostatin-1 inhibited p38 phosphorylation in macrophages. Ferostatin-1 inhibits macrophage ferroptosis, reduces MMP2/9 levels in plaques, and stabilizes the hemorrhagic plaques., Conclusion:  Our results suggested that hemin-induced macrophage ferroptosis promotes p38 pathway activation and MMP2/9 overexpression, which may play a crucial role in increasing hemorrhagic plaque vulnerability. These findings provide insights into the pathogenesis of hemorrhagic plaques and suggest that targeting macrophage ferroptosis may be a promising strategy for stabilizing vulnerable plaque., Competing Interests: None declared., (Thieme. All rights reserved.)

Background: Autophagy, as a regulator of cell survival, plays an important role in atherosclerosis (AS). Sperm associated antigen 5 (SPAG5) is closely associated with the classical autophagy pathway, PI3K/Akt/mTOR signaling pathway. This work attempted to investigate whether SPAG5 can affect AS development by regulating autophagy., Methods: Human umbilical vein endothelial cells (HUVECs) were treated with oxidized-low density lipoprotein (ox-LDL) to induce cell damage. ApoE -/- mice were fed a Western diet to establish an AS mouse model. Haematoxylin and eosin (H&E) staining and Oil Red O staining evaluated the pathological changes and in lipid deposition in aortic tissues. CCK-8 and flow cytometry detected cell proliferation and apoptosis. Immunohistochemistry, Enzyme linked immunosorbent assay, qRT-PCR and western blotting assessed the levels of mRNA and proteins., Results: Ox-LDL treatment elevated SPAG5 expression and the expression of autophagy-related proteins, LC3-I, LC3-II, Beclin-1, and p62, in HUVECs. GFP-LC3 dots were increased in ox-LDL-treated HUVECs and LPS-treated HUVECs. SPAG5 knockdown reversed both ox-LDL and LPS treatment-mediated inhibition of cell proliferation and promotion of apoptosis in HUVECs. SPAG5 silencing further elevated autophagy and repressed the expression of PI3K, p-Akt/Akt, and p-mTOR/mTOR in ox-LDL-treated HUVECs. 3-MA (autophagy inhibitor) treatment reversed SPAG5 silencing-mediated increase of cell proliferation and decrease of apoptosis in ox-LDL-treated HUVECs. In vivo, SPAG5 knockdown reduced atherosclerotic plaques in AS mice through activating autophagy and inhibiting PI3K/Akt/mTOR signaling pathway., Conclusion: This work demonstrated that SPAG5 knockdown alleviated AS development through activating autophagy. Thus, SPAG5 may be a potential target for AS therapy., (© 2024. The Author(s).)

Aims: 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., Methods and Results: 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., Conclusion: Limiting IL1RAP-dependent cytokine signalling pathways in atherosclerotic mice reduces plaque burden and plaque inflammation, potentially by limiting plaque chemokine production., Competing Interests: Conflict of interest: E.J.G., C.G., S.R., and D.L. are employed by and hold stocks or options in Cantargia AB. C.G., S.R., and D.L. are signed as co-inventors on a patent related to anti-IL1RAP monoclonal antibodies. D.E. received reagents related to the current study from Cantargia AB., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background and Aims: Atherosclerosis is the primary underlying cause of myocardial infarction and stroke, which are the major causes of death globally. Heparanase (Hpse) is a pro-inflammatory extracellular matrix degrading enzyme that has been implicated in atherogenesis. However, to date the precise roles of Hpse in atherosclerosis and its mechanisms of action are not well defined. This study aims to provide new insights into the contribution of Hpse in different stages of atherosclerosis in vivo., Methods: We generated Hpse gene-deficient mice on the atherosclerosis-prone apolipoprotein E gene knockout (ApoE -/- ) background to investigate the impact of Hpse gene deficiency on the initiation and progression of atherosclerosis after 6 and 14 weeks high-fat diet feeding, respectively. Atherosclerotic lesion development, blood serum profiles, lesion composition and aortic immune cell populations were evaluated., Results: Hpse-deficient mice exhibited significantly reduced atherosclerotic lesion burden in the aortic sinus and aorta at both time-points, independent of changes in plasma cholesterol levels. A significant reduction in the necrotic core size and an increase in smooth muscle cell content were also observed in advanced atherosclerotic plaques of Hpse-deficient mice. Additionally, Hpse deficiency reduced circulating and aortic levels of VCAM-1 at the initiation and progression stages of disease and circulating MCP-1 levels in the initiation but not progression stage. Moreover, the aortic levels of total leukocytes and dendritic cells in Hpse-deficient ApoE -/- mice were significantly decreased compared to control ApoE -/- mice at both disease stages., Conclusions: This study identifies Hpse as a key pro-inflammatory enzyme driving the initiation and progression of atherosclerosis and highlighting the potential of Hpse inhibitors as novel anti-inflammatory treatments for cardiovascular disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Aims: Diabetic atherosclerotic vascular disease is characterized by extensive vascular calcification. However, an elevated blood glucose level alone does not explain this pathogenesis. We investigated the metabolic markers underlying diabetic atherosclerosis and whether extracellular Hsp90α (eHsp90α) triggers vascular endothelial calcification in this particular metabolic environment., Methods: A parallel human/animal model metabolomics approach was used. We analyzed 40 serum samples collected from 24 patients with atherosclerosis and from the STZ-induced ApoE -/- mouse model. A multivariate statistical analysis of the data was performed, and mouse aortic tissue was collected for the assessment of plaque formation. In vitro, the effects of eHsp90α on endothelial cell calcification were assessed by serum analysis, Western blotting and immunoelectron microscopy., Results: Diabetic ApoE -/- mice showed more severe plaque lesions and calcification damage. Stearamide, oleamide, l-thyroxine, l-homocitrulline and l-citrulline are biomarkers of diabetic ASVD; l-thyroxine was downregulated in both groups, and the thyroid sensitivity index was correlated with serum Hsp90α concentration. In vitro studies showed that eHsp90α increased Runx2 expression in endothelial cells through the LRP1 receptor. l-thyroxine reduced the increase in Runx2 levels caused by eHsp90α and affected the distribution and expression of LRP1 through hydrogen bonding with glutamine at position 1054 in the extracellular segment of LRP1., Conclusions: This study provides a mechanistic link between characteristic serum metabolites and diabetic atherosclerosis and thus offers new insight into the role of extracellular Hsp90α in promoting vascular calcification., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The aim of this study was to elucidate the specific mechanism through which 7-difluoromethoxy-5,4'-dimethoxygenistein (DFMG) inhibits angiogenesis in atherosclerosis (AS) plaques, given its previously observed but poorly understood inhibitory effects. In vitro, a model using Human Umbilical Vein Endothelial (HUVEC-12) cells simulated the initial lesion in the atherosclerotic pathological process, specifically oxidative stress injury, by exposing cells to 30 μmol/L LPC. Additionally, an AS mouse model was developed in ApoE knockout mice through a 16-week period of high-fat feeding. DFMG demonstrated a reduction in tubule quantities in the tube formation assay and neovascularization induced by oxidative stress-damaged endothelial cells in the chicken embryo chorioallantoic membrane assay. Furthermore, DFMG decreased lipid levels in the blood of ApoE knockout mice with AS, along with a decrease in atherosclerotic plaques and neovascularizations in the aortic arch and descending aorta of AS animal models. DFMG treatment upregulated microRNA140 (miR-140) expression and suppressed VEGF secretion in HUVEC-12 cells. These effects were counteracted by Toll-like receptor 4 (TLR4) overexpression in HUVEC-12 cells subjected to oxidative injury or in a mouse model of AS. Dual-luciferase reporter assays demonstrated that miR-140 directly targeted TLR4. Immunohistochemical assay findings indicated a significant inverse relationship between miR-140 expression and TLR4 expression in ApoE knockout mice subjected to a high-fat diet. The study observed a close association between DFMG inhibitory effects on angiogenesis and plaque stability in AS, and the inhibition of the TLR4/NF-κB/VEGF signaling pathway, negatively regulated by miR-140., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Bai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Atherosclerosis (AS) is considered to be one of the main pathogenic factors of coronary heart disease, cerebral infarction and peripheral vascular disease. Oxidative stress and inflammation run through the occurrence and development of atherosclerosis and related cardiovascular events. Muscone is a natural extract of deer musk and also the main physiological active substance of musk. This study investigated the impact of muscone on atherosclerosis. ApoE-/- mice were used to establised AS model and injected with low-dose (4 mg/kg/day) or high-dose (8 mg/kg/day) of muscone intraperitoneally for 4 weeks. Then aortic tissues were collected, and pathological sections of the aorta were prepared for oil red staining, HE and masson staining. The changes of MDA, SOD, VCAM-1, NF-κB, and TNF-α were observed by Western blotting or immunofluorescence staining. The results showed that high-dose muscone could effectively reduce the plaque area/aortic root area and relative atherosclerotic area, reduce the collagen composition in plaque tissue. In addition, we also found that high-dose muscone can effectively increase MDA level, reduce the level of SOD, and inhibit the expression of VCAM-1, NF-κB/p65, TNF-α in arterial plaques. Our results indicate that the administration of muscone has the benefit of inhibiting atherosclerosis. The potential mechanisms may be associated with antioxidant effect and inhibition of inflammatory reaction in arterial plaques. With the increasing understanding of the relationship between muscone and atherosclerosis, muscone has high potential value as a new drug to treat atherosclerosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: The occurrence and development of atherosclerosis, a common chronic inflammatory vascular disease, are closely related to cardiovascular and cerebrovascular diseases. Banxia Baizhu Tianma Decoction (BBTD) is a representative traditional Chinese medicine formula that resolves phlegm, disperses wind, invigorates the spleen and eliminates dampness and is also a commonly used clinical medication for treating vascular diseases., Aim of the Study: To explore the pharmacological mechanisms of BBTD in alleviating atherosclerosis, the present study was carried out by conducting an integrative analysis of aortic and perivascular adipose tissue (PVAT) proteomics and metabolomics., Materials and Methods: Eight-week-old ApoE-/- mice were randomly divided into the BBTD group and the model group, and nine age-matched C57BL/6J (C57) mice were used as the control group (n = 9). The C57 mice were fed a standard diet, while the ApoE -/- mice were fed a high-fat, high-cholesterol diet for 12 weeks. Mice in the BBTD group were transgastrically administered BBTD at a dose of 17.8 g/kg/day for 8 weeks, while the model group and control group mice received an equivalent volume of saline by gavage. Histomorphology of the aortas and PVAT was assessed by HE staining, oil red O staining, Masson staining, and α-SMA and CD68 immunohistochemical methods. An integrative analysis of aortic proteomics, PVAT proteomics and PVAT metabolomics was conducted to study the pharmacological mechanisms of BBTD., Results: Compared to the model group, mice treated with BBTD had thicker fibrous caps, increased collagen content, less erosion of smooth muscle cells and infiltration of macrophages, as well as a relatively low inflammatory response level, suggesting that BBTD treatment reduced plaque vulnerability. Omics analysis suggested that BBTD treatment demonstrated anti-atherosclerotic effects and increased plaque stability in the aorta by activating the TGF-beta pathway. Simultaneously, BBTD inhibited PVAT inflammation levels (decreased the levels of MCP and IL-6). Proteomics and metabolomics of PVAT suggested that the targets of BBTD included upregulation of the α-linolenic acid metabolic pathway and downregulation of multiple inflammatory pathways, such as the NF-kappa B signalling pathway, primary immunodeficiency and Th17 cell differentiation in PVAT., Conclusions: BBTD reduces the vulnerability of atherosclerotic plaques and inhibits the inflammatory phenotype of perivascular adipose tissue., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Inflammatory bowel disease (IBD) and atherosclerosis (AS) are both common chronic inflammatory diseases with similar pathophysiological mechanisms. Some studies have shown that IBD patients are at increased risk for early atherosclerosis, myocardial infarction and venous thrombosis. Here we set up a hybrid mouse model associated with atherosclerosis and acute colitis in order to investigate the interplay of the two diseases. We fed ApoE-/- mice with high fat diet to establish atherosclerosis model, and used animal ultrasound machine to detect the artery of mice noninvasively. Then a new hybrid model of atherosclerosis and acute colitis was prepared by drinking water for 7 days. At the end of the experiment, the hybrid model mice showed typically pathological and intuitionistic changes of atherosclerosis and acute colitis. We found the shortened colon length, high histopathological scores of the colon with mucosal erosion and necrosis, hyperlipidemia, a plaque-covered mouse aorta and plaque with foam cells and lipid deposition in the hybrid model group, which proved that the hybrid model was successfully established. At the same time, ultrasonic detection showed that the end-diastolic blood flow velocity and the relative dilation value were decreased, while systolic time / diastolic time, the wall thickness, systolic diameters as well as diastolic diameters were gradually increased, and statistical significance appeared as early as 8 weeks. We clearly described the process of establishing a hybrid model of atherosclerosis and acute colitis, which might provide a repeatable platform for the interaction mechanism exploring and drug screening of atherosclerosis and inflammatory bowel disease in preclinical study., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Atherosclerosis(AS) poses a pressing challenge in contemporary medicine. Formononetin (FMN) plays a crucial role in its prevention and treatment. However, the detailed impact of FMN on the stability of atherosclerotic plaques and its underlying mechanisms remain to be elucidated., Methods: An intervention consisting of FMN was given along with a high-fat food regimen in the ApoE-/- mouse model. The investigation included the evaluation of the degree of atherosclerotic lesion, the main components of the plaque, lipid profiles, particular markers indicating M1/M2 macrophage phenotypes, the quantities of factors related to inflammation, the infiltration of macrophages, and the identification of markers linked to the α7nAChR/JAK2/STAT3 axis effect molecules., Results: The evaluation of aortic morphology in ApoE-/-mice revealed that FMN significantly improved the plaque area, fibrous cap protrusion, lipid deposition, and structural alterations on the aortic surface, among other markers of atherosclerosis,and there is concentration dependence. Furthermore, the lipid content of mouse serum was assessed, and the results showed that the low-, medium-, and high-dosage FMN groups had significantly lower levels of LDL-C, ox-LDL, TC, and TG. The results of immunohistochemical staining indicated that the low-, medium-, and high-dose FMN therapy groups had enhanced CD206 expression and decreased expression of CD68 and iNOS. According to RT-qPCR data, FMN intervention has the potential to suppress the expression of iNOS, COX-2, miR-155-5p, IL-6, and IL-1β mRNA, while promoting the expression of IL-10, SHIP1, and Arg-1 mRNA levels. However, the degree of inhibition varied among dosage groups. Western blot investigation of JAK/STAT signaling pathway proteins and cholinergic α7nAChR protein showed that p-JAK2 and p-STAT3 protein expression was suppressed at all dosages, whereas α7nAChR protein expression was enhanced., Conclusions: According to the aforementioned findings, FMN can reduce inflammation and atherosclerosis by influencing macrophage polarization, blocking the JAK/STAT signaling pathway, and increasing α7nAChR expression., (© 2024. The Author(s).)

Background: According to former research, the atherosclerotic plaque is thought to be aggravated by intraplaque neovessels (IPN) and intraplaque hemorrhage (IPH). Intriguingly, a lower incidence of IPH was found in plaque treated with melatonin. In this study, we attempted to investigate the impact and underlying mechanism regarding the influences of melatonin upon IPN., Methods: A mouse model was established by subjecting the high fat diet (HFD)-fed ApoE-/- mice to tandem stenosis (TS) surgery with melatonin and GW9662, a PPARγ antagonist, being given by gavage. In vitro experiment was conducted with HUVECs exposing to according treatments of VEGF, melatonin, GW9662, or Y27632., Results: Plaque and IPN were attenuated by treatment with melatonin, which was then reversed by blocking PPARγ. Western blotting results showed that melatonin increased PPARγ and decreased RhoA/ROCK signaling in carotid artery. Elevated RhoA/ROCK signaling was observed in melatonin-treated mice when PPARγ was blocked. In accordance with it, experiments using protein and mRNA from HUVECs revealed that melatonin inhibited the RhoA/ROCK signaling by enhancing PPARγ. According to in vitro study, melatonin was able to inhibit cell migration and angiogenesis, which was aborted by GW9662. Blockage of ROCK using Y27632 was able to cease the effect of GW9662 and restored the suppression on cell migration and angiogenesis by melatonin., Conclusions: Our study demonstrates that melatonin is able to curb development of plaque and IPN formation by inhibiting the migration of endothelial cells via PPARγ- RhoA-ROCK pathway. That provides a therapeutic potential for both melatonin and PPARγ agonist targeting IPN, IPH, and atherosclerotic plaque., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: Xinmaikang (XMK) tablets, a Chinese patent medicine, have been used for the prevention and treatment of atherosclerosis (AS) clinically. However, the underlying mechanism of XMK is far from completely illustrated., Aim of the Study: This study aimed to determine whether XMK alleviates AS in Apolipoprotein E-knockout (ApoE-/-) mice and to explore the potential mechanism of action in bone marrow-derived macrophages (BMDMs)., Materials and Methods: XMK decoction was analyzed by an LC‒MS/MS assay. Molecular docking was conducted to determine the interaction of XMK molecular ligands and AS targets. In vivo, 10 ApoE-/- mice were selected as the control group. Fifty ApoE-/- mice were randomly divided into 5 groups: the model group, low-, medium-, and high-dose XMK groups and the simvastatin group. Mice in the control group were fed a chow diet, and the other 5 groups were fed a high-fat diet (HFD) for 12 weeks. After 12 weeks, the treatment groups were administered low-dose XMK (2.28·kg-1·d), medium-dose XMK (4.55·kg-1·d), high-dose XMK (9.1 kg -1  d) and simvastatin (91 mg -1  d) for another 12 weeks. Serum enzymology assays tested AST/ALT, Cr, LDH and CK-MB levels. The atherosclerotic plaques and lipid deposition were measured by Oil red O (ORO) staining and Hematoxylin and Eosin (H&E) staining. Then, we examined the body weight and serum lipids (TC, TG, LDL-C and HDL-C) of the mice. ELISA was performed to determine the levels of inflammatory factors (IL-6, TNF-ɑ, VCAM-1, CXCL8 and CCL2). SREBP2/NLRP3 signaling pathway-related genes (SREBP2, NLRP3, ASC, IL-1β and Caspase-1) were analyzed by RT‒qPCR and western blotting. In vitro, LPS-stimulated BMDMs were treated with different concentrations of XMK (1, 2.5, 5, 10, 20, and 40 μg/ml). Immunofluorescence staining (SREBP2, NLRP3), adenovirus infection and siRNA knockdown (SREBP2, NLRP3, Caspase-1 and ASC) were conducted as complements to the in vivo experiment., Results: Molecular docking showed a stable interaction between the effective components of XMK and SREBP2 and NLRP3. Serum enzymology assays revealed the medication safety of XMK in cardiac, hepatic and renal function. Studies in vivo indicated that XMK improved serum lipids (TC, TG, LDL-C and HDL-C) and reduced plaque area. Body weight decreased, and the expression of inflammatory cytokines (IL-6, TNF-ɑ and VCAM-1) was inhibited. Then, XMK downregulated the mRNA and protein expression of SREBP2, NLRP3, ASC, IL-1β and Caspase-1. In vitro, the above findings were reinforced in BMDMs, and knocking down SREBP2 restrained the effect of XMK on the NLRP3/ASC/Caspase-1 signaling pathway., Conclusions: XMK restrains AS by improving inflammation through the SREBP2-mediated NLRP3/ASC/Caspase-1 signaling pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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

Ethnopharmacological Relevance: Dengyinnaotong (DYNT) is a traditional Chinese medicine-based patent drug officially approved for the treatment of ischemic stroke primarily based on its indigenous application for the treatment of cardiovascular and cerebrovascular diseases in Southwest China. Atherosclerosis is the principal pathology underlying the pathogenesis of ischemic stroke and coronary artery disease. However, whether DYNT is effective at mitigating atherosclerosis remains unknown., Aims of the Study: The purpose of the current study is to evaluate the potential impact of DYNT treatment on the atherosclerotic lesions and associated pathological mechanisms., Materials and Methods: Histological, immunohistochemical, molecular biological approaches were adopted to investigate the pharmacological impact of DYNT treatment on atherosclerosis and associated pathophysiological alterations in the high fat diet (HFD)-fed ApoE gene deficient (ApoE -/- ) mice., Results: DYNT treatment reduced the size of the atherosclerotic plaques, alleviated the necrotic core, lowered the lipid retention, mitigated the macrophagic burden and decreased the expression of proatherogenic chemokine Ccl2 in the atherosclerotic lesions. DYNT treatment also offered partial protection against atherogenic dyslipidemia and mitigated hepatic lipid content as well as fatty liver pathologies in the HFD-fed ApoE -/- mice. Furthermore, DYNT treatment protected against atherosclerosis-associated gut dysbiosis and impairment in the intestinal epithelial barrier., Conclusions: Our work provides novel preclinical evidence that underpins the multifaceted effects of DYNT in the control of atherosclerosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Atherosclerosis (AS) is a chronic inflammatory disease involving cell death and inflammatory responses. Pyroptosis, a newly discovered pro-inflammatory programmed cell death process, exacerbates inflammatory responses. However, the roles of cathepsin B (CTSB) in pyroptosis and AS remain unclear. To gain further insight, we fed ApoE-/- mice a high-fat diet to investigate the effects and mechanisms of CTSB overexpression and silencing on AS. We also explored the specific role of CTSB in vascular smooth muscle cells (VSMCs) in vitro. The study revealed that high-fat diet led to the formation of AS plaques, and CTSB was found to increase the AS plaque lesion area. Immunohistochemical and TUNEL/caspase-1 staining revealed the existence of pyroptosis in atherosclerotic plaques, particularly in VSMCs. In vitro studies, including Hoechst 33342/propidium iodide staining, a lactate dehydrogenase (LDH) release assay, detection of protein indicators of pyroptosis, and detection of interleukin-1β (IL-1β) in cell culture medium, demonstrated that oxidized low-density lipoprotein (ox-LDL) induced VSMC pyroptosis. Additionally, CTSB promoted VSMC pyroptosis. Ox-LDL increased the expression of CTSB, which in turn activated the NOD-like receptor protein 3 (NLRP3) inflammasome and promoted NLRP3 expression by facilitating nuclear factor kappa B (NF-κB) p65 nuclear translocation. This effect could be attenuated by the NF-κB inhibitor SN50. Our research found that CTSB not only promotes VSMC pyroptosis by activating the NLRP3 inflammasome, but also increases the expression of NLRP3., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: It has been observed previously that chronic methamphetamine (METH) administration could upregulate neuropeptide Y (NPY) expression and promote atherosclerotic formation in apolipoprotein E knockout (ApoE-/-) mice fed with a normal cholesterol or high diet and NPY might be involved in the pathogenesis of METHinduced atherogenic effects through NPY Y1 receptor pathway. Vulnerable coronary atherosclerotic plaque (VP) is a critical pathological finding responsible for the acute coronary syndrome (ACS). In this study, we explored whether METH abuse could aggravate the formation of VP in ApoE-/- mice fed with high cholesterol diet., Objective: The purpose of this study was to observe if chronic METH administration could aggravate vulnerable plaque (VP) formation in ApoE-/- mice fed with a highcholesterol diet., Methods: Male ApoE-/- mice fed with a high-cholesterol diet were intraperitoneally injected with normal saline (NS) or 8 mg/kg/day METH (M8) for 24 weeks. Body weight was monitored from baseline to 24 weeks at 2 weeks intervals. After 24 weeks of treatment, plasma lipid variables were measured. Movat's staining and immunohistochemical staining were performed on frozen sections of the aortic roots to calculate VP percentage and intraplaque hemorrhage (IPH) percentage and detect expression of NPY, vascular endothelial growth factor (VEGF), and CD31. In vitro, the expressions of Y2R, VEGF, and CD31 were detected by immunofluorescence staining in aortic endothelial cells incubated with PBS, 100μM METH, 10nmol NPY, or 100μM METH plus 10nmol NPY for 12 hours., Results: The CD31 positive area, percentage of IPH, VP, and the expressions of NPY and VEGF were significantly increased in the M8 group than in the NS group. In vitro, the expressions of Y2R, VEGF, and CD31 were significantly increased in the METH+NPY group than in the PBS, METH, and NPY groups and these effects could be blunted by treatment with a Y2R antagonist or DPPIV inhibitor., Conclusion: Chronic METH administration could aggravate VP in ApoE-/- mice fed with a high-cholesterol diet, possibly through upregulating vascular NPY and VEGF expression and promoting angiogenesis and vessel rupture in atherosclerotic plaques. Our findings indicated that increased VP formation might contribute to the development of acute coronary syndrome post-chronic METH abuse by activating DPPIV/NPY/Y2R pathway., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

In this study, the significance of oxidized low-density lipoprotein (ox-LDL) in promoting the progression of atherosclerosis was investigated by inducing the differentiation of macrophages into the M2 subtype within a high-fat diet-induced ApoE -/- mouse model. The study also evaluated the effects of β2-AR agonists and blockers on this process. Ox-LDL was found to have significantly promoted the differentiation of macrophages into the M2 type and induced related functional alterations. Furthermore, it activated the pyroptosis pathway and encouraged the release of lactate dehydrogenase. The administration of β2-AR agonists intensified these processes, while β2-AR blockers had the opposite effect. In animal experiments, the model group displayed elevated numbers of M2-type macrophages beneath the aortic root intima, an increased rate of plaque destruction, and the formation of atherosclerotic plaques compared to the control group. The SAL (Salbutamol) group exhibited even more severe plaque development than the model group. Conversely, the ICI (ICI118551) group demonstrated M2-type macrophage levels comparable to the control group, with a higher plaque destruction rate than controls but significantly lower than the model group, and no atherosclerotic plaques. These findings suggest that ox-LDL promoted the differentiation of recruited monocytes into M2-type macrophages, leading to a shift in the inflammatory response from M1 to M2 macrophages. This alteration resulted in the persistence of atherosclerotic inflammation, as M2-type macrophages were prone to cell membrane rupture (such as pyroptosis), contributing to the continuous recruitment of circulating monocytes and heightened inflammatory reactions within atherosclerotic plaques. Consequently, this process fueled the progression of atherosclerosis.

Background: The role of complement component 1q (C1Q) related genes on human atherosclerotic plaques (HAP) is less known. Our aim is to establish C1Q associated hub genes using single-cell RNA sequencing (scRNA-seq) and bulk RNA analysis to diagnose and predict HAP patients more effectively and investigate the association between C1Q and HAP (ischemic stroke) using bidirectional Mendelian randomization (MR) analysis., Methods: HAP scRNA-seq and bulk-RNA data were download from the Gene Expression Omnibus (GEO) database. The C1Q-related hub genes was screened using the GBM, LASSO and XGBoost algorithms. We built machine learning models to diagnose and distinguish between types of atherosclerosis using generalized linear models and receiver operating characteristics (ROC) analyses. Further, we scored the HALLMARK&#95;COMPLEMENT signaling pathway using ssGSEA and confirmed hub gene expression through qRT-PCR in RAW264.7 macrophages and apoE-/- mice. Furthermore, the risk association between C1Q and HAP was assessed through bidirectional MR analysis, with C1Q as exposure and ischemic stroke (IS, large artery atherosclerosis) as outcomes. Inverse variance weighting (IVW) was used as the main method., Results: We utilized scRNA-seq dataset (GSE159677) to identify 24 cell clusters and 12 cell types, and revealed seven C1Q associated DEGs in both the scRNA-seq and GEO datasets. We then used GBM, LASSO and XGBoost to select C1QA and C1QC from the seven DEGs. Our findings indicated that both training and validation cohorts had satisfactory diagnostic accuracy for identifying patients with HPAs. Additionally, we confirmed SPI1 as a potential TF responsible for regulating the two hub genes in HAP. Our analysis further revealed that the HALLMARK&#95;COMPLEMENT signaling pathway was correlated and activated with C1QA and C1QC. We confirmed high expression levels of C1QA, C1QC and SPI1 in ox-LDL-treated RAW264.7 macrophages and apoE-/- mice using qPCR. The results of MR indicated that there was a positive association between the genetic risk of C1Q and IS, as evidenced by an odds ratio (OR) of 1.118 (95%CI: 1.013-1.234, P = 0.027)., Conclusion: The authors have effectively developed and validated a novel diagnostic signature comprising two genes for HAP, while MR analysis has provided evidence supporting a favorable association of C1Q on IS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cui, Tang, Gao, Li, Zhang and Li.)

Background: Clinical observation suggests the atheroprotective effect of chloroquine and its derivatives, while its mechanism remains unclear. This study aimed to observe the protective effect of chloroquine against atherosclerosis and explore the underlying mechanism., Methods: Ataxia telangiectasia mutated (ATM) wild-type or haploinsufficient apolipoprotein-E-knockout (ATM +/+ ApoE -/- or ATM +/- ApoE -/- ) mice were treated with different dosages of chloroquine. Anti-CD25 antibody was used to deplete natural Tregs in ATM +/+ ApoE -/- mice. The atherosclerotic burden in different groups of mice was comprehensively evaluated by H&E staining and Masson staining. The effect of chloroquine on the regulatory T cells (Tregs) was assessed in vivo and in vitro by flow cytometry and immunohistochemical staining. The expression of related proteins was detected by real-time polymerase chain reaction and western blotting., Results: In ATM +/+ ApoE -/- mice, chloroquine alleviated atherosclerotic lesions, stabilized the plaque, and increased Treg counts in the atherosclerotic lesions and spleens. However, in ATM haploinsufficient mice (ATM +/- ApoE -/- ), chloroquine no longer prevented atherosclerosis or impacted Treg counts. Abolishing Treg cells using an anti-CD25 antibody in vivo abrogated the atheroprotective effect of chloroquine. In vitro, chloroquine promoted the differentiation of Tregs from naïve T cells, which was accompanied by enhanced ATM/AMP-activated protein kinase (AMPK) activity and reduced downstream mammalian target of rapamycin (mTOR) activity., Discussion: These findings suggest that chloroquine ameliorates atherosclerosis and stabilizes plaque by modulating Tregs differentiation through the regulation of the ATM/AMPK/mTOR pathway., Competing Interests: The authors declare no conflicts of interest in this work., (Thieme. All rights reserved.)

Ethnopharmacological Relevance: QiShenYiQi pill (QSYQ), a Chinese compound medicine, originate from BuYangHuanWu decoction in the Qing dynasty, and has been used to treat ischemic cardiovascular diseases for more than two hundred years in China. Multi-central randomized double-blind controlled studies have proved that QSYQ has similar efficacy as enteric coated aspirin in the secondary prevention of myocardial infarction., Aim of Study: The aim of study was to explore the effect of QSYQ on reverse cholesterol transport (RCT) pathway during atherosclerosis., Materials and Methods: Eight-week-old male apoE -/- mice (on the gene background of C57BL/6J) were fed with a high-fat western diet and treated with low dose and high dose of QSYQ, as well as the positive control agent, liver X receptor-α (LXR-α) agonist GW3965. Eight weeks later, mice were sacrificed and the aorta was collected for atherosclerotic analysis. The aortic root was stained with Oil red O to evaluate the area of atherosclerotic lesion, and stained with immunohistochemistry to analyze the intra-plaque component and RCT protein in atherosclerotic plaque. The thoracic aorta was used to detect differentially expressed genes by comparative transcriptome RNA-seq and the protein expression of RCT pathway by western blotting., Results: After eight weeks of treatment, we found that both of QSYQ and LXR-α agonist reduced atherosclerotic plaque area significantly, and decreased the intra-plaque component, including the lipid, the smooth muscle cell and the macrophage. Compared with the control group, there were 49 differentially expressed genes in low-dose QSYQ group, including 21 up-regulated genes and 28 down-regulated genes. The results of GO and KEGG analysis showed that the differentially expressed genes mainly concentrated in the negative regulation of lipid biosynthesis, positive regulation of lipid metabolism, cell response to lipids, negative regulation of lipid storage, fatty acid degradation, and glycerol ester metabolism. Both of QSYQ and LXR-α agonist reduced the protein expression of CD36 and increased the protein expression of PPARγ-LXRα/β-ABCA1 in atherosclerotic plaque., Conclusion: The anti-atherosclerotic mechanism of QSYQ was involved in inhibiting lipid phagocytosis and promoting reverse cholesterol transport, therefore reducing lipid deposition and inflammatory cells in plaque., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Aims: Atherosclerosis is driven by multiple processes across multiple body systems. For example, the innate immune system drives both atherogenesis and plaque rupture via inflammation, while coronary artery-occluding thrombi formed by the coagulation system cause myocardial infarction and death. However, the interplay between these systems during atherogenesis is understudied. We recently showed that coagulation and immunity are fundamentally linked by the activation of interleukin-1α (IL-1α) by thrombin, and generated a novel knock-in mouse in which thrombin cannot activate endogenous IL-1α [IL-1α thrombin mutant (IL-1αTM)]., Methods and Results: Here, we show significantly reduced atherosclerotic plaque formation in IL-1αTM/Apoe-/- mice compared with Apoe-/- and reduced T-cell infiltration. However, IL-1αTM/Apoe-/- plaques have reduced vascular smooth muscle cells, collagen, and fibrous caps, indicative of a more unstable phenotype. Interestingly, the reduced atherogenesis seen with thrombin inhibition was absent in IL-1αTM/Apoe-/- mice, suggesting that thrombin inhibitors can affect atherosclerosis via reduced IL-1α activation. Finally, bone marrow chimeras show that thrombin-activated IL-1α is derived from both vessel wall and myeloid cells., Conclusions: Together, we reveal that the atherogenic effect of ongoing coagulation is, in part, mediated via thrombin cleavage of IL-1α. This not only highlights the importance of interplay between systems during disease and the potential for therapeutically targeting IL-1α and/or thrombin, but also forewarns that IL-1 may have a role in plaque stabilization., Competing Interests: Conflict of interest: None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Abstract: Many studies have confirmed that macrophage autophagy injury negatively impacts the pathogenesis of atherosclerosis (AS). Meanwhile, the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway affects AS progression by regulating macrophage autophagy. We previously reported that the herbal formula San Jie Tong Mai Fang (SJTMF) elicits lipid regulatory and anti-inflammatory properties. Hence, the current study used an ApoE -/- high-fat diet-fed mouse model to determine whether SJTMF elicits protective effects against AS progression by means of the regulation of macrophage autophagy through the PI3K/AKT/mTOR signaling pathway. Our results show that SJTMF reduced the number of atherosclerotic plaques, foam cell formation, and intimal thickness in mouse aorta. In addition, SJTMF improved blood lipid metabolism and inflammatory levels in mice. We also observed that SJTMF caused macrophages to be polarized toward the M2 phenotype through the inhibition of the PI3K/AKT/mTOR signaling pathway. In addition, the abundances of LC3-II/I and beclin1 proteins-key autophagy molecules-were increased, whereas that of p62 was decreased, resulting in the promotion of macrophage autophagy. Taken together, these findings indicate that SJTMF may regulate the polarization of macrophages by inhibiting the PI3K/AKT/mTOR signaling pathway, thereby reducing atherosclerotic plaque damage in ApoE -/- mice, thereby promoting macrophage autophagy and eliciting a significant antiarteriosclerosis effect. Hence, SJTMF may represent a promising new candidate drug for the treatment of AS., Competing Interests: The authors report no conflicts of interest., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background: The ADAMTS7 locus was genome-wide significantly associated with coronary artery disease. Lack of the ECM (extracellular matrix) protease ADAMTS-7 (A disintegrin and metalloproteinase-7) was shown to reduce atherosclerotic plaque formation. Here, we sought to identify molecular mechanisms and downstream targets of ADAMTS-7 mediating the risk of atherosclerosis., Methods: Targets of ADAMTS-7 were identified by high-resolution mass spectrometry of atherosclerotic plaques from Apoe -/- and Apoe -/- Adamts7 -/- mice. ECM proteins were identified using solubility profiling. Putative targets were validated using immunofluorescence, in vitro degradation assays, coimmunoprecipitation, and Förster resonance energy transfer-based protein-protein interaction assays. ADAMTS7 expression was measured in fibrous caps of human carotid artery plaques., Results: In humans, ADAMTS7 expression was higher in caps of unstable as compared to stable carotid plaques. Compared to Apoe -/- mice, atherosclerotic aortas of Apoe -/- mice lacking Adamts-7 (Apoe -/- Adamts7 -/- ) contained higher protein levels of Timp-1 (tissue inhibitor of metalloprotease-1). In coimmunoprecipitation experiments, the catalytic domain of ADAMTS-7 bound to TIMP-1, which was degraded in the presence of ADAMTS-7 in vitro. ADAMTS-7 reduced the inhibitory capacity of TIMP-1 at its canonical target MMP-9 (matrix metalloprotease-9). As a downstream mechanism, we investigated collagen content in plaques of Apoe -/- and Apoe -/- Adamts7 -/- mice after a Western diet. Picrosirius red staining of the aortic root revealed less collagen as a readout of higher MMP-9 activity in Apoe -/- as compared to Apoe -/- Adamts7 -/- mice. To facilitate high-throughput screening for ADAMTS-7 inhibitors with the aim of decreasing TIMP-1 degradation, we designed a Förster resonance energy transfer-based assay targeting the ADAMTS-7 catalytic site., Conclusions: ADAMTS-7, which is induced in unstable atherosclerotic plaques, decreases TIMP-1 stability reducing its inhibitory effect on MMP-9, which is known to promote collagen degradation and is likewise associated with coronary artery disease. Disrupting the interaction of ADAMTS-7 and TIMP-1 might be a strategy to increase collagen content and plaque stability for the reduction of atherosclerosis-related events., Competing Interests: Disclosures Dr Schunkert has received personal fees from MSD SHARP & DOHME, AMGEN, Bayer Vital GmbH, Boehringer Ingelheim, Daiichi-Sankyo, Novartis, Servier, Brahms, Bristol-Myers-Squibb, Medtronic, Sanofi Aventis, Synlab, Pfizer, and Vifor T as well as grants and personal fees from Astra-Zeneca outside the submitted work. Drs Schunkert and Kessler are named inventors on a patent application for prevention of restenosis after angioplasty and stent implantation outside the submitted work. Dr Kessler received lecture fees from Bayer, Bristol-Myers Squibb, Abbott, and Astra-Zeneca, which are unrelated to this work. Dr Bernhagen is an inventor on patents related to anti-MIF and antichemokine strategies in inflammation and CVD (cardiovascular diseases) outside the submitted work. The other authors report no conflicts.

Background: Atherosclerosis (AS) is still the major cause of cardiovascular disease (CVD) as well as stroke. Endothelial metabolic disorder has been found to be activated and then promote endothelial cells (ECs) injury, which is regarded to initiate AS progression. N-acetylneuraminic acid (Neu5Ac), a metabolite produced by hexosamine-sialic acid pathway branching from glucose metabolism, was presented as a notable biomarker of CVD and is positively correlated with ECs function. However, few studies explain whether Neu5Ac regulate AS progression by affecting EC function as well as its involved mechanisms are still unknown. Methods: Here, we mimicked an animal model in ApoE -/- mice which displaying similar plasma Neu5Ac levels with AS model to investigate its effect on AS progression. Results: We found that Neu5Ac exacerbated plaques area and increased lipids in plasma in absence of HFD feeding, and ECs inflammatory injury was supposed as the triggering factor upon Neu5Ac treatment with increasing expression of IL-1β, ICAM-1, and promoting ability of monocyte adhesion to ECs. Mechanistic studies showed that Neu5Ac facilitated SLC3A2 binding to ubiquitin and then triggered P62 mediated degradation, further leading to accumulation of lipid peroxidation in ECs. Fer-1 could inhibit ECs injury and reverse AS progression induced by Neu5Ac in ApoE -/- mice. Interestingly, mitochondrial dysfunction was also partly participated in ECs injury after Neu5Ac treatment and been reversed by Fer-1. Conclusions: Together, our study unveils a new mechanism by which evaluated metabolite Neu5Ac could promote SLC3A2 associated endothelial ferroptosis to activate ECs injury and AS plaque progression, thus providing a new insight into the role of Neu5Ac-ferroptosis pathway in AS. Also, our research revealed that pharmacological inhibition of ferroptosis may provide a novel therapeutic strategy for premature AS., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Aims: Chemoattractants and their cognate receptors are essential for leucocyte recruitment during atherogenesis, and atherosclerotic plaques preferentially occur at predilection sites of the arterial wall with disturbed flow (d-flow). In profiling the endothelial expression of atypical chemoattractant receptors (ACKRs), we found that Ackr5 (CCRL2) was up-regulated in an endothelial subpopulation by atherosclerotic stimulation. We therefore investigated the role of CCRL2 and its ligand chemerin in atherosclerosis and the underlying mechanism., Methods and Results: By analysing scRNA-seq data of the left carotid artery under d-flow and scRNA-seq datasets GSE131776 of ApoE-/- mice from the Gene Expression Omnibus database, we found that CCRL2 was up-regulated in one subpopulation of endothelial cells in response to d-flow stimulation and atherosclerosis. Using CCRL2-/-ApoE-/- mice, we showed that CCRL2 deficiency protected against plaque formation primarily in the d-flow areas of the aortic arch in ApoE-/- mice fed high-fat diet. Disturbed flow induced the expression of vascular endothelial CCRL2, recruiting chemerin, which caused leucocyte adhesion to the endothelium. Surprisingly, instead of binding to monocytic CMKLR1, chemerin was found to activate β2 integrin, enhancing ERK1/2 phosphorylation and monocyte adhesion. Moreover, chemerin was found to have protein disulfide isomerase-like enzymatic activity, which was responsible for the interaction of chemerin with β2 integrin, as identified by a Di-E-GSSG assay and a proximity ligation assay. For clinical relevance, relatively high serum levels of chemerin were found in patients with acute atherothrombotic stroke compared to healthy individuals., Conclusions: Our findings indicate that d-flow-induced CCRL2 promotes atherosclerotic plaque formation via a novel CCRL2-chemerin-β2 integrin axis, providing potential targets for the prevention or therapeutic intervention of atherosclerosis., Competing Interests: Conflict of interest: None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aims: The variant p.Arg149Cys in ACTA2, which encodes smooth muscle cell (SMC)-specific α-actin, predisposes to thoracic aortic disease and early onset coronary artery disease in individuals without cardiovascular risk factors. This study investigated how this variant drives increased atherosclerosis., Methods and Results: Apoe-/- mice with and without the variant were fed a high-fat diet for 12 weeks, followed by evaluation of atherosclerotic plaque formation and single-cell transcriptomics analysis. SMCs explanted from Acta2R149C/+ and wildtype (WT) ascending aortas were used to investigate atherosclerosis-associated SMC phenotypic modulation. Hyperlipidemic Acta2R149C/+Apoe-/- mice have a 2.5-fold increase in atherosclerotic plaque burden compared to Apoe-/- mice with no differences in serum lipid levels. At the cellular level, misfolding of the R149C α-actin activates heat shock factor 1, which increases endogenous cholesterol biosynthesis and intracellular cholesterol levels through increased HMG-CoA reductase (HMG-CoAR) expression and activity. The increased cellular cholesterol in Acta2R149C/+ SMCs induces endoplasmic reticulum stress and activates PERK-ATF4-KLF4 signaling to drive atherosclerosis-associated phenotypic modulation in the absence of exogenous cholesterol, while WT cells require higher levels of exogenous cholesterol to drive phenotypic modulation. Treatment with the HMG-CoAR inhibitor pravastatin successfully reverses the increased atherosclerotic plaque burden in Acta2R149C/+Apoe-/- mice., Conclusion: These data establish a novel mechanism by which a pathogenic missense variant in a smooth muscle-specific contractile protein predisposes to atherosclerosis in individuals without hypercholesterolemia or other risk factors. The results emphasize the role of increased intracellular cholesterol levels in driving SMC phenotypic modulation and atherosclerotic plaque burden., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Atherosclerosis is the root cause of major cardiovascular diseases (CVD) such as myocardial infarction and stroke. ADP-ribosylation factor 6 (Arf6) is a ubiquitously expressed GTPase known to be involved in inflammation, vascular permeability and is sensitive to changes in shear stress. Here, using atheroprone, ApoE-/- mice, with a single allele deletion of Arf6 (HET) or wildtype Arf6 (WT), we demonstrate that reduction in Arf6 attenuates atherosclerotic plaque burden and severity. We found that plaque burden in the descending aorta was lower in HET compared to WT mice (p˂0.001) after the consumption of an atherogenic Paigen diet for 5 weeks. Likewise, luminal occlusion, necrotic core size, plaque grade, elastic lamina breaks, and matrix deposition were lower in the aortic root atheromas of HET compared to WT mice (all p≤0.05). We also induced advanced human-like complex atherosclerotic plaque in the left carotid artery using partial carotid ligation surgery and found that atheroma area, plaque grade, intimal necrosis, intraplaque hemorrhage, thrombosis, and calcification were lower in HET compared to WT mice (all p≤0.04). Our findings suggest that the atheroprotection afforded by Arf6 heterozygosity may result from reduced immune cell migration (all p≤0.005) as well as endothelial and vascular smooth muscle cell proliferation (both p≤0.001) but independent of changes in circulating lipids (all p≥0.40). These findings demonstrate a critical role for Arf6 in the development and severity of atherosclerosis and suggest that Arf6 inhibition can be explored as a novel therapeutic strategy for the treatment of atherosclerotic CVD., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Gogulamudi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Stabilizing and inhibiting plaque formation is a key challenge for preventing and treating ischemic stroke. KDM1A-mediated histone modifications, which involved in the development of training immunity, ultimately exacerbate the outcomes of inflammation. Although lncRNAs can recruit KDM1A to participate in histone methylation modification and regulate inflammation, cell proliferation, and other biological processes, little is known about the role of KDM1A-lncRNA interaction during atherosclerosis. The present study sought to delineate the effect of the interaction between lnc&#95;000048 and KDM1A on plaque rupture in carotid atherosclerosis, as well as the potential mechanism. Our results revealed that lnc&#95;000048 reduced the activity of histone demethylase and activated MAP2K2 expression by interacting with KDM1A. Furthermore, upregulated lnc&#95;000048 indirectly regulated ERK phosphorylation by MAP2K2 and eventually activated the inflammatory response through the MAPK pathway, which was involved in atherosclerosis. Importantly, our study using ApoE-/- mice confirmed the regulatory role of lnc&#95;000048 in promoting inflammation and collagen degradation in atherosclerotic plaques. These results suggest that targeting the lnc&#95;000048 /KDM1A/MAP2K2/ERK axis may be a promising strategy for preventing atherosclerosis., (© 2023. The Author(s).)