Mouse Model Of Atherosclerosis Research Articles

Role of STING Deficiency in Amelioration of Mouse Models of Lupus and Atherosclerosis.

Systemic lupus erythematosus (SLE) is a systemic autoimmune syndrome characterized by autoreactive responses to nucleic acids, dysregulation of the type I interferon (IFN-I) pathway, and accelerated atherosclerosis. The stimulator of IFN genes (STING), a cytosolic DNA sensor, has pathogenic implications in various inflammatory diseases. However, its specific role in SLE pathogenesis, particularly in tissue damage, remains unclear. This study aimed to elucidate the role of STING in murine models of Toll-like receptor 7 (TLR7)-driven lupus and atherosclerosis. A TLR7-driven lupus model was induced using imiquimod (IMQ) in wild-type (WT) and STING knockout (Sting1-/-) mice on a B6 background. Mice were assessed for organ involvement, serum autoantibodies, and innate and adaptive immune responses. Additionally, Sting1-/- mice were backcrossed to apolipoprotein E knockout (Apoe-/-) mice, and both Apoe-/- and Apoe-/-Sting1-/- mice were fed a high-fat chow diet to induce atherosclerosis. Phenotypic assessments were conducted. Compared with IMQ-treated WT mice, Sting1-/- mice exhibited reduced disease severity in the lupus-like phenotype, characterized by decreased splenomegaly, lower renal immune complex deposition and renal damage, diminished expansion of myeloid cells, and reduced activation of T and B lymphocytes. IMQ-induced DNA release associated with IFN-β production and subsequent IFN-induced responses were attenuated in Sting1-/- mice. DNase I treatment mitigated IMQ-induced proinflammatory responses in WT mice but had no effect in Sting1-/- mice. Furthermore, STING deficiency conferred protection against vascular damage and reduced atherosclerosis burden, accompanied by decreased IFN-I production. Human monocyte-derived macrophages treated with IFN-I significantly internalized more acetylated low-density lipoprotein when compared with untreated cells, whereas an association between oxidized nucleic acids and disease activity and vascular damage was found in human SLE. These findings highlight a pathogenic role of STING and downstream IFN responses in TLR7-driven autoimmunity, vascular damage and atherosclerosis, supporting a therapeutic potential for STING inhibition in SLE treatment. Further research is warranted to elucidate the mechanisms underlying STING's involvement in these processes and to explore the feasibility of targeting STING as a therapeutic strategy in SLE and related autoimmune disorders.

Taoren Honghua Decoction alleviates atherosclerosis by inducing autophagy and inhibiting the PI3K-AKT signaling pathway to regulate cholesterol efflux and inflammatory responses

BackgroundTaoren Honghua Decoction (THD) is a traditional Chinese formula known for enhancing blood circulation and demonstrating clinical efficacy in the treatment of cardiovascular and cerebrovascular diseases. However, the primary active components and the underlying mechanisms by which THD exerts its therapeutic effects on atherosclerosis (AS) remain insufficiently characterized. ObjectiveThis study aims to systematically validate the protective effects of THD on AS and elucidate its potential molecular mechanisms through an integrative approach involving network pharmacology, in vivo, and in vitro experiments. MethodsThe main active ingredients and corresponding targets of all traditional Chinese medicines in THD were collected from the TCSMP and BATMAN-TCM databases. Potential targets of AS were identified using the OMIM, DrugBank, DisGeNET, and CTD databases, and AS microarray gene data were obtained from the GEO database. A drug active ingredient-target relationship network and a PPI network were constructed using Cytoscape 3.9.2 software. The molecular functions of the core targets were annotated through GO and KEGG enrichment analyses to further elucidate the potential molecular mechanisms of THD’s anti-AS effects. The ApoE−/−mouse AS model was constructed through a high-fat diet (HFD), and RAW264.7 macrophage model was induced with ox-LDL to further validate the results of network pharmacology. ResultsNetwork pharmacology analysis revealed that the main five active ingredients of THD include quercetin, apigenin, luteolin, kaempferol, and tanshinone IIA. Subsequently, by analyzing the intersection genes of the main active ingredient targets of THD and the AS targets, a total of ten core targets were identified: TP53, PPARG, JUN, AKT1, INS, IL6, SIRT1, TNF, ESR1, and STAT3. These are considered the core targets of THD in the treatment of AS. The GO and KEGG enrichment analysis results indicate that THD may exert anti-AS effects by regulating lipid metabolism and the PI3K-AKT signaling pathway. In vivo and in vitro experiments showed that THD reduced circulating lipid levels, decreased intraplaque lipid accumulation, and increased intraplaque collagen fiber content in HFD-induced ApoE−/− mice. Additionally, THD reduced ox-LDL-induced macrophage-derived foam cell formation, inhibited the expression of inflammatory factors IL-6 and TNF-α, and promoted the expression of cholesterol efflux regulatory proteins PPARγ, ABCA1, and ABCG1. Notably, the autophagy inhibitor 3-MA reversed these effects, confirming that THD’s action involves autophagy activation, evidenced by increased LC3II/I and decreased p62 levels. ConclusionThis study demonstrates that THD exerts significant anti-AS effects through the inhibition of the PI3K/AKT signaling pathway and the activation of autophagy, thereby promoting cholesterol efflux and mitigating inflammation. By integrating network pharmacology with experimental validation, these findings provide a comprehensive understanding of THD’s mechanisms in treating AS and offer a solid theoretical basis for its potential clinical application.

Endothelial Rho kinase controls blood vessel integrity and angiogenesis.

The Rho kinases 1 and 2 (ROCK1/2) are serine-threonine specific protein kinases that control actin cytoskeleton dynamics. They are expressed in all cells throughout the body, including cardiomyocytes, smooth muscle cells and endothelial cells, and intimately involved in cardiovascular health and disease. Pharmacological ROCK inhibition is beneficial in mouse models of hypertension, atherosclerosis, and neointimal thickening that display overactivated ROCK. However, the consequences of endothelial ROCK signaling deficiency in vivo remain unknown. To address this issue, we analyzed endothelial cell (EC) specific ROCK1 and 2 deletions. We generated Cdh5-CreERT2 driven, tamoxifen inducible loss of function alleles of ROCK1 and ROCK2 and analyzed mouse survival and vascular defects through cellular, biochemical, and molecular biology approaches. We observed that postnatal or adult loss of endothelial ROCK1 and 2 was lethal within a week. Mice succumbed to multi-organ hemorrhage that occurred because of loss of vascular integrity. ECs displayed deficient cytoskeletal actin polymerization that prevented focal adhesion formation and disrupted junctional integrity. Retinal sprouting angiogenesis was also perturbed, as sprouting vessels exhibited lack of polymerized actin and defective lumen formation. In a three-dimensional endothelial sprouting assay, combined knockdown of ROCK1/2 or knockdown or ROCK2 but not ROCK1 led to reduced sprouting, lumenization and cell polarization defects caused by defective actin and altered VE-cadherin dynamics. The isoform specific role of endothelial ROCK2 correlated with ROCK2 substrate specificity for FAK and LIMK. By analyzing single and three allele mutants we show that one intact allele of ROCK2 is sufficient to maintain vascular integrity in vivo . Endothelial ROCK1 and 2 maintain junctional integrity and ensure proper angiogenesis and lumen formation. The presence of one allele of ROCK2 is sufficient to maintain vascular growth and integrity. These data indicate the need of careful consideration for the use of ROCK inhibitors in disease settings.

Increased caveolin 1 by human antigen R exacerbates Porphyromonas gingivali-induced atherosclerosis by modulating oxidative stress and inflammatory responses

Objective: Many different types of infectious oral diseases have been identified clinically, including chronic periodontitis. Porphyromonas gingivalis is the main pathogen causing chronic periodontitis, which is closely related to atherosclerosis (AS) and can promote the expression levels of caveolin 1 (Cav-1) and induced ribonucleic acid (RNA)-binding protein human antigen R (HuR). However, the roles of Cav-1 and its relationship with HuR in P. gingivalis-mediated AS progression remain largely unknown. Here, we aimed to detect the role and molecular mechanisms of Cav-1 in P. gingivalis-mediated AS. Material and Methods: To investigate the role of Cav-1 in P. gingivalis-mediated AS, we infected human umbilical vein endothelial cells (HUVECs) with P. gingivalis at a multiplicity of infection of 100:1 for 6, 12, and 24 h to simulate P. gingivalis-induced AS models in vitro and then transfected them with Cav-1 small interfering RNA to silence Cav-1. Combining molecular biology experimental techniques such as cell counting kit-8 assay, enzyme-linked immunosorbent assay, immunofluorescence staining, flow cytometry, Western blotting, and Oil Red O staining, and apolipoprotein E-deficient AS model mice, the impacts of Cav-1 on cell viability, inflammation, oxidative stress, apoptosis, Cav-1 and intercellular cell adhesion molecule-1 (ICAM-1) levels, and atherosclerotic plaque formation were investigated. Then, the relationship between Cav-1 and HuR was investigated through biotin pull-down and RNA immunoprecipitation assays, reverse transcription quantitative polymerase chain reaction, and Western blot. Results: P. gingivalis can induce Cav-1 expression in a time- and dose-dependent manner (P < 0.05). This effect can inhibit the proliferation of HUVECs (P < 0.05). Cav-1 interference repressed inflammatory response, reactive oxygen species (ROS) and ICAM-1 levels, and apoptosis in the HUVECs (P < 0.05). Cav-1 messenger RNA was stabilized by HuR, which can bind to the 3’ untranslated region of Cav-1. Increase in HuR level reversed the effects of Cav-1 silencing on ROS and ICAM-1 levels and apoptosis in the HUVECs (P < 0.05). In addition, the levels of inflammatory response, oxidative stress, and atherosclerotic plaque formation induced by P. gingivalis in the mouse model were significantly reduced after Cav-1 expression was inhibited (P < 0.05). Conclusion: HuR-activated Cav-1 may promote atherosclerotic plaque formation by modulating inflammatory response and oxidative stress, leading to AS.

Toxicity and efficacy study of a combination of two retinoic acids in an ApoE knockout mouse model of atherosclerosis.

Atherosclerosis is a major contributor to cardiovascular disease, characterized by inflammation and lipid accumulation in arterial walls, leading to plaque formation. Elevated low-density lipoprotein cholesterol is a primary risk factor for atherosclerosis. All-trans retinoic acid (ATRA), a metabolite of vitamin A, has demonstrated anti-inflammatory effects and potential in regulating vascular injury. 9-cisretinoic acid (9cRA) is an active metabolite of vitamin A and activates the retinoid X receptor. This study investigates whether potassium retinoate (PA9RA), a synthetic combination of ATRA and 9cRA, offers superior efficacy in treating atherosclerosis compared to established treatments such as clopidogrel and atorvastatin. Male ApoE-/- mice were fed a Western-type diet and treated with PA9RA, clopidogrel, or atorvastatin for 10 weeks. The body weight, organ weight, serum biochemistry, and histopathology, including atherosclerotic lesion area and liver steatosis were assessed. PA9RA treatment led to a significant reduction in body weight and inguinal fat, with the 45 mg/kg/day dose showing marked efficacy in decreasing atherosclerotic lesion size and ameliorating liver steatosis. Histopathological evaluation revealed decreased foam cell formation and improved liver histology in PA9RA-treated groups compared to controls. Notable side effects included epidermal hyperplasia and gastric hyperplasia at high doses of PA9RA. PA9RA exhibits superior efficacy over clopidogrel and atorvastatin in ameliorating atherosclerosis and fatty liver in ApoE-/- mice. This study highlights PA9RA's potential as a promising therapeutic agent for atherosclerosis. Further research is needed to elucidate its mechanisms of action and assess long-term safety and efficacy.

Abstract 4113617: Single-cell Analysis Reveals Endothelial Cell CD45 Deficiency Prevents Endothelial-to-Mesenchymal Transition (EndMT) in Atherosclerosis

Introduction: Protein-tyrosine-phosphatase CD45 is exclusively expressed in all nucleated cells of the hematopoietic system but is rarely expressed in endothelial cells (ECs). However, a recent study indicated that activation of the endogenous CD45 promoter in human endothelial colony forming cells induced expression of EndMT marker genes. Also, the CD45 phosphatase inhibitor blocked EndMT activities in TGFβ1-treated ovine mitral valve endothelial cells. EndMT contributes to atherosclerotic pathobiology and is associated with more complex plaques. We identified CD45-positive ECs undergoing EndMT in atherosclerotic ApoE-deficient (ApoE -/- ) mice. Here, we aim to investigate whether endothelial CD45-specific deletion can prevent EndMT in a mouse model of atherosclerosis. Methods and Results: We generated a tamoxifen-inducible EC-specific CD45-deficient mouse strain (EC-iCD45KO) on an ApoE -/- background (C57BL/6) and fed these mice a Western diet for 16 weeks. We isolated and enriched mouse aortic endothelial cells with CD31 beads to perform single-cell RNA-seq. Cells populated 15 major clusters after integrating the single-cell transcriptomic profiles. In the EC-iCD45KO group, the population of endothelial, resident macrophage, and mesenchymal-endothelial transition cells increased while vascular smooth muscle, EndMT, and fibroblast cells decreased. In EndMT cells, EC markers (Cdh5, Cldn5, Pecam1) increased, but SMC markers (Acta2) and EndMT markers (Col1a2 and Col3a1）decreased. In addition, we characterized EndMT cells into two sub-clusters: 1 and 2. EC-specific CD45 deletions reduced the cell numbers in the EndMT2 sub-cluster in atherosclerotic mice but had less impact on the EndMT1 sub-cluster. FGFR pathways were potentiated while TGFβ1 and Tgfbr2 decreased in EndMT1 sub-cluster， and the genes controlling endothelial cell development were upregulated but the genes controlling actin cytoskeleton organization were downregulated in both EndMT1 and 2 sub-clusters. Conclusion: Our single-cell analysis indicates that the loss of endothelial CD45 protects against EndMT-driven atherosclerosis, inhibiting TGFβ and EndoMT marker expression while stimulating FGFR pathways. Consistently, EC-iCD45KO/ApoE -/- mice showed reduced plaque macrophages, expression of cell adhesion molecules, foam cell formation, and lesion development when compared to ApoE -/- controls. Thus, targeting endothelial CD45 may be a novel therapeutic strategy for EndMT and atherosclerosis.

Abstract 4138304: Macrophage ZIP8 Enhances Efferocytosis and Mitigates Atherosclerosis by Modulating cellular Manganese (Mn) Homeostasis

Introduction: ZIP8, also known as SLC39A8, is a transmembrane transporter responsible for the movement of various divalent metal ions. Multiple genome-wide association studies (GWAS) have revealed a significant association between a mutation in ZIP8 (rs13107325; Ala391Thr) and cardiovascular diseases. While its physiologic role is incompletely understood, recent studies implicate that Zn2+ influx into monocytes regulated by ZIP8 is a novel factor determining their adhesion and recruitment to atherosclerotic lesions. The potential role of ZIP8 in modulating macrophage function and thereby influencing the development of atherosclerosis warrants further investigation. Methods and Results: Using an ApoE -/- ZIP8 A393T missense mutation mouse model, we quantified atherosclerotic plaque burden and lipid profiles following induction of atherosclerosis. The presence of the ZIP8 A393T missense mutation resulted in increased necrotic regions and plaque load in the atherosclerosis mouse model, while having no impact on total cholesterol (TC) or low-density lipoprotein cholesterol (LDL-C) levels. Confocal microscopy and flow cytometry to investigate how reduced ZIP8 expression affects macrophage efferocytosis. When siRNA was used to knock down ZIP8 in vitro, macrophage efferocytosis was reduced. Subsequently, Macrophage-specific ZIP8 knockout mice (ZIP8 LysM ) demonstrated a decrease in the efferocytosis index in primary macrophage. Additionally, in vivo thymic and peritoneal efferocytosis assays showed an impaired efferocytosis phenotype in ZIP8 LysM mice. Confocal microscopy showed ZIP8 LysM mice have no effect on macrophage binding to apoptotic cells. The lack of or incompleteness of the F-ACTIN ring formed by macrophages surrounding apoptosis in the ZIP8 LysM mice, and the prolonged duration of phagocytosis ring formation suggests that the internalization process is compromised. The role of ZIP8 ion transport in the efferocytosis impairment mechanism was also investigated.The regulation of macrophage efferocytosis via the metabolism of D-galactose, glutamine, glutamate, and Zn 2+ is unaffected by ZIP8. Mn 2+ was identified as a vital regulatory factor in the efferocytosis function of ZIP8 in macrophages. Conclusions: ZIP8 influences the efferocytotic capacity of macrophages and regulates the clearance of apoptotic cells through Mn 2+ , suggesting that ZIP8 and Mn 2+ hold promise as a key therapeutic targets for the treatment of atherosclerotic cardiovascular diseases.

Histone demethylase JMJD1C advances macrophage foam cell formation and atherosclerosis progression by promoting the transcription of PCSK9.

Macrophage is considered as a critical driving factor in the progression of atherosclerosis (AS), and epigenetic heterogeneity contributes important mechanisms in this process. Here, we identified that a histone demethylase jumonji domain-containing protein 1C (JMJD1C) is a promising biomarker for atherosclerotic cerebral infarction through clinical analysis. Then, AOPE-/- mice fed with a high fat diet and RAW264.7 cells induced by oxidized low-density lipoprotein (ox-LDL) were used as AS models to verify the function of JMJD1C in AS development in vivo and in vitro. JMJD1C knockdown significantly reduced plaque area, inflammation and endothelial damage in AS model mice, and also alleviated foam cell formation, inflammatory cytokines production and cell apoptosis in ox-LDL-treated RAW264.7 cells. Mechanistically, JMJD1C promoted the transcription of proprotein convertase subtilisin/kexin type 9 (PCSK9) through mediating H3 Lysine 9 demethylation. The effects of JMJD1C knockdown on ox-LDL-induced macrophages were blocked by PCSK9 overexpression. Altogether, our study proves that JMJD1C advances macrophage foam cell formation, inflammation and apoptosis to accelerate AS progression through H3 demethylation of PCSK9. The findings underscore the important role of JMJD1C-mediated histone modification in macrophage regulation and AS progression, which brings a new insight into the pathobiology of AS.

Aryl-hydrocarbon receptor in smooth muscle cells protect against dioxin induced adverse remodeling of atherosclerosis.

Environmental exposure to dioxin has been linked to increased myocardial infarction. Smooth muscle cells (SMC) in the coronary vasculature play a critical role in atherosclerotic plaque remodeling due to their phenotypic plasticity, however, the detailed mechanism linking dioxin exposure to adverse SMC modulation is not well understood. Single-cell RNA and ATAC sequencing and histological analyses were performed on the aorta from mouse models of atherosclerosis exposed to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) or control. Primary human coronary artery SMC (HCASMC) treated in culture with TCDD were used to perform RNA-Seq, ATAC-Seq, and functional phenotypic assays. ChIP-Seq was performed with antibodies against Aryl-hydrocarbon receptor (AHR) and TCF21, two of known SMC modulating transcription factors. Modulated SMC were the most transcriptionally responsive cell type to dioxin in the atherosclerotic aorta. Dioxin accelerated disease phenotype by promoting a modulated SMC phenotype early, resulting in increased lesion size, migration of SMC, and macrophage recruitment to the lesion. We found C3 expressing modulated SMCs to be likely contributing to the increased macrophage recruitment and inflammation. Analysis of the RNA-Seq data from HCASMC treated with TCDD showed differential enrichment of biological pathways related to cell migration, localization, and inflammation. Furthermore, ATAC-Seq data showed a significant activation for pathways regulating vascular development, cell migration, inflammation, and apoptosis. With TCDD treatment, there was also enrichment of AHR ChIP-Seq peaks, while the TCF21 enrichment decreased significantly. The SMC-specific Ahr knockout resulted in increased oxidative stress in SMC, increased lesion size and macrophage content, and loss of SMC lineage cells in the lesion cap when exposed to TCDD, consistent with a more vulnerable plaque phenotype. Dioxin adversely remodels atherosclerotic plaque by accelerating the SMC- phenotypic modulation, and increasing inflammation and oxidative stress resulting in increased macrophage recruitment and lesion size. Dioxin may adversely affect the SMC phenotype and disease state by affecting the TCF21 occupancy in the open chromatin regions. Furthermore, we observed that SMC-specific deletion of Ahr in mice resulted in worsening of dioxin mediated SMC modulation and atherosclerosis, suggesting that Ahr in SMC confers protection against dioxin by promoting a stable plaque phenotype and reducing dioxin induced oxidative stress. Exposure to dioxin, an environmental pollutant present in tobacco smoke and air pollution, accelerates smooth muscle cell modulation, and atherosclerosis.Dioxin exposure leads to inflammatory smooth muscle cell phenotype characterized by complement pathway activation and increased macrophage recruitment to plaqueAryl-hydrocarbon receptor in SMC protects against oxidative stress, and promotes a stable plaque phenotype.

Kindlin-2 Phase Separation in Response to Flow Controls Vascular Stability.

Atheroprotective shear stress preserves endothelial barrier function, while atheroprone shear stress enhances endothelial permeability. Yet, the underlying mechanisms through which distinct flow patterns regulate EC integrity remain to be clarified. This study aimed to investigate the involvement of Kindlin-2, a key component of focal adhesion and endothelial adherens junctions crucial for regulating endothelial cell (EC) integrity and vascular stability. Mouse models of atherosclerosis in EC-specific Kindlin-2 knockout mice (Kindlin-2iΔEC) were used to study the role of Kindlin-2 in atherogenesis. Pulsatile shear (2±4 dynes/cm2) or oscillatory shear (0.5±4 dynes/cm2) were applied to culture ECs. Live-cell imaging, fluorescence recovery after photobleaching assay, and optoDroplet assay were used to study the liquid-liquid phase separation (LLPS) of Kindlin-2. Co-immunoprecipitation, mutagenesis, proximity ligation assay, and transendothelial electrical resistance assay were used to explore the underlying mechanism of flow-regulated Kindlin-2 function. We found that Kindlin-2 localization is altered under different flow patterns. Kindlin-2iΔEC mice showed heightened vascular permeability. Kindlin-2iΔEC were bred onto ApoE-/- mice to generate Kindlin-2iΔEC; ApoE-/- mice, which displayed a significant increase in atherosclerosis lesions. In vitro data showed that in ECs, Kindlin-2 underwent LLPS, a critical process for proper focal adhesion assembly, maturation, and junction formation. Mass spectrometry analysis revealed that oscillatory shear increased arginine methylation of Kindlin-2, catalyzed by PRMT5 (protein arginine methyltransferase 5). Functionally, arginine hypermethylation inhibits Kindlin-2 LLPS, impairing focal adhesion assembly and junction maturation. Notably, we identified R290 of Kindlin-2 as a crucial residue for LLPS and a key site for arginine methylation. Finally, pharmacologically inhibiting arginine methylation reduces EC activation and plaque formation. Collectively, our study elucidates that mechanical force induces arginine methylation of Kindlin-2, thereby regulating vascular stability through its impact on Kindlin-2 LLPS. Targeting Kindlin-2 arginine methylation emerges as a promising hemodynamic-based strategy for treating vascular disorders and atherosclerosis. URL: https://www.clinicaltrials.gov; Unique identifier: NCT02783300.

Aspirin inhibits atherosclerosis through macrophage pyroptosis by interacting with Lactobacillus johnsonii and its metabolite butyrate

Abstract Introduction Atherosclerotic cardiovascular disease (ASCVD) is a chronic progressive disease closely related to metabolism and inflammation. Aspirin inhibits cyclooxygenase (COX) and has antiplatelet aggregation effects, considered as the cornerstone of ASCVD treatment. Numerous studies have shown that gut microbiota (GM) and its metabolites affects host metabolism. Studies have also shown that people taking aspirin exhibit specific changes in the microbiota profile. However, whether the interaction of aspirin with GM and its metabolites affects the therapeutic efficacy of aspirin in treating atherosclerosis (AS) have not been reported. Purpose This study aimed to explore the interaction of aspirin with the GM and its metabolites in the treatment of AS, to reveal the specific mechanism of the GM-metabolite axis and provide potential new targets for the combined treatment of AS. Methods To establish the mouse model of atherosclerosis by high-fat diet, and the atherosclerotic plaque burden and composition were analyzed. The role of GM in aspirin anti-atherosclerosis was explored by antibiotics and fecal microbiota transplantation (FMT). The third generation 16S RNA sequencing was used to explore the effects of aspirin on species diversity and abundance of GM, and to screen differential microorganisms for intervention of AS mice. UHPLC-MS and GC-MS were used to explore the changes of intestinal metabolites induced by aspirin. The correlation between gut microbiota and metabolites was also investigated. Finally, in vitro and in vivo experiments were performed to confirm the effects and mechanisms of metabolites on macrophage pyroptosis and atherosclerosis. Results Aspirin has anti-atherosclerosis effect. After removing gut microbiota, the therapeutic effect of aspirin on atherosclerosis was decreased. Three generations of 16S rRNA gene sequencing results showed that aspirin supplementation alters the diversity of gut microbiota, and increased the relative abundance of Lactobacillus johnsonii(L.johnsonii) and Ligilactobacillus murinus(L.murinus). After gavage of L.johnsonii and L.murinus, the plaque burden and macrophage pyroptosis were significantly reduced in AS mice. Colonization with L.johnsonii and L.murinus significantly increased the abundance of probiotics and butyrate level in the feces of AS mice and L.johnsonii and L.murinus were positively correlated with butyrate. Supplementation of butyrate inhibited macrophage pyroptosis and atherosclerosis in vivo. In vitro, butyrate could inhibit the expression of NLRP3/Caspase-1/IL-1β and pyroptosome production, and this process mainly by activating butyrate receptors GPR41, GPR43 and GPR109A. Conclusions The gut microbiota may mediate the anti-AS effect of aspirin by promoting an increase in the abundance of L.johnsonii and L.murinus. Butyrate produced by Lactobacillus may mediate the anti-AS effect of aspirin, and inhibiting macrophage pyroptosis and the progression of AS.

Visualizing Immune Checkpoint Inhibitors Derived Inflammation in Atherosclerosis.

Immune checkpoint inhibitor (ICI) usage has resulted in immune-related adverse events in patients with cancer, such as accelerated atherosclerosis. Of immune cells involved in atherosclerosis, the role of CCR2+ (CC motif chemokine receptor 2-positive) proinflammatory macrophages is well documented. However, there is no noninvasive approach to determine the changes of these cells in vivo following ICI treatment and explore the underlying mechanisms of immune-related adverse events. Herein, we aim to use a CCR2 (CC motif chemokine receptor 2)-targeted radiotracer and positron emission tomography (PET) to assess the aggravated inflammatory response caused by ICI treatment in mouse atherosclerosis models and explore the mechanism of immune-related adverse events. Apoe-/- mice and Ldlr-/- mice were treated with an ICI, anti-PD1 (programmed cell death protein 1) antibody, and compared with those injected with either isotype control IgG or saline. The radiotracer 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-ECL1i (extracellular loop 1 inverso) was used for PET imaging of CCR2+ macrophages. Atherosclerotic arteries were collected for molecular characterization. CCR2 PET revealed significantly higher radiotracer uptake in both Apoe-/- and Ldlr-/- mice treated with anti-PD1 compared with the control groups. The increased expression of CCR2+ cells in Apoe-/- and Ldlr-/- mice was confirmed by immunostaining and flow cytometry. Single-cell RNA sequencing revealed elevated expression of CCR2 in myeloid cells. Mechanistically, IFNγ (interferon gamma) was essential for aggravated inflammation and atherosclerotic plaque progression following anti-PD1 treatment. Accelerated atherosclerotic plaque inflammation triggered by anti-PD1 treatment can be noninvasively detected by 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-ECL1i PET. Aggravated plaque inflammation is time- and dose-dependent and predominately mediated by IFNγ signaling. This study warrants further investigation of CCR2 PET as a noninvasive approach to visualize atherosclerotic plaque inflammation and explore the underlying mechanism following ICI treatment.

High expression of PLA2G2A in fibroblasts plays a crucial role in the early progression of carotid atherosclerosis

BackgroundIn mouse models of atherosclerosis, knockout of the PLA2G2A gene has been shown to reduce the volume of atherosclerotic plaques. Clinical trials have demonstrated the potential of using the sPLA2 inhibitor Varespladib in combination with statins to reduce lipid levels. However, this approach has not yielded the expected results in reducing the risk of cardiovascular events. Therefore, it is necessary to further investigate the mechanisms of PLA2G2A.MethodsSingle-cell transcriptome data from two sets of carotid plaques, combined with clinical patient information. were used to describe the expression characteristics of PLA2G2A in carotid plaques at different stages. In order to explore the mechanisms of PLA2G2A, we conducted enrichment analysis, cell–cell communication analysis and single-cell regulatory network inference and clustering analyses. We validated the above findings at the cellular level.ResultsOur findings indicate that PLA2G2A is primarily expressed in vascular fibroblasts and shows significant cell interactions with macrophages in the early-stage, especially in complement and inflammation-related pathways. We also found that serum sPLA2 levels have stronger diagnostic value in patients with mild carotid artery stenosis. Subsequent comparisons of single-cell transcriptomic data from early and late-stage carotid artery plaques corroborated these findings and predicted transcription factors that might regulate the progression of early carotid atherosclerosis (CA) and the expression of PLA2G2A.ConclusionsOur study discovered and validated that PLA2G2A is highly expressed by vascular fibroblasts and promotes plaque progression through the activation of macrophage complement and coagulation cascade pathways in the early-stage of CA.

Integrated metabolomics and network pharmacology to reveal the mechanisms of Shexiang Baoxin pill against atherosclerosis

BackgroundAtherosclerosis is a disease marked by the development of lipid lesions within the endothelium and continues to be a prominent contributor to global mortality. Shexiang Baoxin pill (SBP) has been employed in the management of numerous cardiovascular diseases, but the complex mechanisms by which it operates remain obscure. This research was conducted to determine the potential impact of SBP on atherosclerosis and the underlying regulatory mechanism involved. MethodNetwork pharmacology was utilized to predict the key drug-disease targets, and a nontargeted metabolomic assay was applied to identify the key metabolites and metabolic pathways. A mouse atherosclerosis model was constructed to clarify the protective effect of SBP on atherosclerosis, and in vivo and in vitro tests were performed to verify the analysis results and clarify the mechanism through which SBP affects atherosclerosis. ResultsThe results show that SBP can exert a protective effect in vivo by decreasing lipid levels, plaque formation and endothelial damage. Network pharmacology and metabolomics revealed that MAPK3, AKT1 and STAT3 were the hub targets and that trimethylamine n-oxide (TMAO) was the pivotal metabolite. Due to the atherogenic effect of TMAO, the corresponding protective effect of SBP was investigated in vitro. SBP inhibited TMAO-induced endothelial cell apoptosis and oxidative stress and counteracted the upregulation of MAPK3, AKT1, and STAT3 expression. Molecular docking and enzymatic inhibition suggested that the active components of SBP could bind stably to key target proteins. ConclusionTaken together, based on the integrated metabolomics and network pharmacology, our findings suggest that SBP may be implicated in TMAO-induced atherosclerosis by affecting endothelial function and bile acid synthesis. We observed that SBP may ameliorate atherosclerosis by regulating TMAO levels through multiple pathways, which may provide a novel direction and insight for SBP involved in cardiovascular protection by mediating the gut-heart axis.

Anti-Inflammatory Oxysterol, Oxy210, Inhibits Atherosclerosis in Hyperlipidemic Mice and Inflammatory Responses of Vascular Cells.

We previously reported that Oxy210, an oxysterol-based drug candidate, exhibits antifibrotic and anti-inflammatory properties. We also showed that, in mice, it ameliorates hepatic hallmarks of non-alcoholic steatohepatitis (NASH), including inflammation and fibrosis, and reduces adipose tissue inflammation. Here, we aim to investigate the effects of Oxy210 on atherosclerosis, an inflammatory disease of the large arteries that is linked to NASH in epidemiologic studies, shares many of the same risk factors, and is the major cause of mortality in people with NASH. Oxy210 was studied in vivo in APOE*3-Leiden.CETP mice, a humanized mouse model for both NASH and atherosclerosis, in which symptoms are induced by consumption of a high fat, high cholesterol "Western" diet (WD). Oxy210 was also studied in vitro using two cell types that are important in atherogenesis: human aortic endothelial cells (HAECs) and macrophages treated with atherogenic and inflammatory agents. Oxy210 reduced atherosclerotic lesion formation by more than 50% in hyperlipidemic mice fed the WD for 16 weeks. This was accompanied by reduced plasma cholesterol levels and reduced macrophages in lesions. In HAECs and macrophages, Oxy210 reduced the expression of key inflammatory markers associated with atherosclerosis, including interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), chemokine (C-C motif) ligand 2 (CCL2), vascular cell adhesion molecule-1 (VCAM-1), and E-Selectin. In addition, cholesterol efflux was significantly enhanced in macrophages treated with Oxy210. These findings suggest that Oxy210 could be a drug candidate for targeting both NASH and atherosclerosis, as well as chronic inflammation associated with the manifestations of metabolic syndrome.

Wall Shear Stress (WSS) Analysis in Atherosclerosis in Partial Ligated Apolipoprotein E Knockout Mouse Model through Computational Fluid Dynamics (CFD).

Atherosclerosis involves an inflammatory response due to plaque formation within the arteries, which can lead to ischemic stroke and heart disease. It is one of the leading causes of death worldwide, with various contributing factors such as hyperlipidemia, hypertension, obesity, diabetes, and smoking. Wall shear stress (WSS) is also known as a contributing factor of the formation of atherosclerotic plaques. Since the causes of atherosclerosis cannot be attributed to a single factor, clearly understanding the mechanisms and causes of its occurrence is crucial for preventing the disease and developing effective treatment strategies. To better understand atherosclerosis and define the correlation between various contributing factors, computational fluid dynamics (CFD) analysis is primarily used. CFD simulates WSS, the frictional force caused by blood flow on the vessel wall with various hemodynamic changes. Using apolipoprotein E knockout (ApoE-KO) mice subjected to partial ligation and a high-fat diet at 1-week, 2-week, and 4-week intervals as an atherosclerosis model, CFD analysis was conducted along with the reconstruction of carotid artery blood flow via magnetic resonance imaging (MRI) and compared to the inflammatory factors and pathological staining. In this experiment, a comparative analysis of the effects of high WSS and low WSS was conducted by comparing the standard deviation of time-averaged wall shear stress (TAWSS) at each point within the vessel wall. As a novel approach, the standard deviation of TAWSS within the vessel was analyzed with the staining results and pathological features. Since the onset of atherosclerosis cannot be explained by a single factor, the aim was to find the correlation between the thickness of atherosclerotic plaques and inflammatory factors through standard deviation analysis. As a result, the gap between low WSS and high WSS widened as the interval between weeks in the atherosclerosis mouse model increased. This finding not only linked the occurrence of atherosclerosis to WSS differences but also provided a connection to the causes of vulnerable plaques.

Simultaneously blocking ANGPTL3 and IL-1β for the treatment of atherosclerosis through lipid-lowering and anti-inflammation.

Blood lipid levels play a critical role in the progression of atherosclerosis. However, even with adequate lipid reduction, significant residual cardiovascular risk remains. Therefore, it is necessary to seek novel therapeutic strategies for atherosclerosis that can not only lower lipid levels but also inhibit inflammation simultaneously. The fusion protein FD03-IL-1Ra was designed by linking the Angiopoietin-like 3 (ANGPTL3) nanobody and human interleukin-1 receptor antagonist (IL-1Ra) sequences to a mutated human immunoglobulin gamma 1 (IgG1) Fc. This construct was transfected into HEK293 cells for expression. The purity and thermal stability of the fusion protein were assessed using SDS-PAGE, SEC-HPLC, and differential scanning calorimetry. Binding affinities of the fusion protein to ANGPTL3 and IL-1 receptor were measured using Biacore T200. The biological activity of the fusion protein was validated through in vitro experiments. The therapeutic efficacy of the fusion protein was evaluated in an ApoE-/- mouse model of atherosclerosis, including serum lipid level determination, histological analysis of aorta and aortic sinus sections, and detection of inflammatory and oxidative stress markers. ImageJ software was utilized for quantitative image analysis. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post hoc test. The FD03-IL-1Ra fusion protein was successfully expressed, with no polymer formation detected, and it demonstrated good thermal and conformational stability. High affinity for both murine and human ANGPTL3 was exhibited by FD03-IL-1Ra, and it was able to antagonize hANGPTL3's inhibition of LPL activity. FD03-IL-1Ra also showed high affinity for both murine and human IL-1R, inhibiting IL-6 expression in A549 cells induced by IL-1β stimulation, as well as suppressing IL-1β-induced activity inhibition in A375.S2 cells. Our study revealed that the fusion protein effectively lowered serum lipid levels and alleviated inflammatory responses in mice. Furthermore, the fusion protein enhanced plaque stability by increasing collagen content within atherosclerotic plaques. These findings highlighted the potential of bifunctional interleukin-1 receptor antagonist and ANGPTL3 antibody fusion proteins for ameliorating the progression of atherosclerosis, presenting a promising novel therapeutic approach targeting both inflammation and lipid levels.

Fluorescence and photoacoustic (FL/PA) dual-modal probe: Responsive to reactive oxygen species (ROS) for atherosclerotic plaque imaging

Accurate and early detection of atherosclerosis (AS) is imperative for their effective treatment. However, fluorescence probes for efficient diagnosis of AS often encounter insufficient deep tissue penetration, which hinders the reliable assessment of plaque vulnerability. In this work, a reactive oxygen species (ROS) activated near-infrared (NIR) fluorescence and photoacoustic (FL/PA) dual model probe TPA-QO-B is developed by conjugating two chromophores (TPA-QI and O–OH) and ROS-specific group phenylboronic acid ester. The incorporation of ROS-specific group not only induces blue shift in absorbance, but also inhibits the ICT process of TPA-QO-OH, resulting an ignorable initial FL/PA signal. ROS triggers the convertion of TPA-QO-B to TPA-QO-OH, resulting in the concurrent amplification of FL/PA signal. The exceptional selectivity of TPA-QO-B towards ROS makes it effectively distinguish AS mice from the healthy. The NIR emission can achieve a tissue penetration imaging depth of 0.3 cm. Moreover, its PA775 signal possesses the capability to penetrate tissues up to a thickness of 0.8 cm, ensuring deep in vivo imaging of AS model mice in early stage. The ROS-triggered FL/PA dual signal amplification strategy improves the accuracy and addresses the deep tissue penetration problem simultaneously, providing a promising tool for in vivo tracking biomarkers in life science and preclinical applications.

LRG1 promotes atherosclerosis by inducing macrophage M1-like polarization

Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by the accumulation of cholesterol-rich lipoproteins in macrophages. How macrophages commit to proinflammatory polarization under atherosclerosis conditions is not clear. Report here that the level of a circulating protein, leucine-rich alpha-2 glycoprotein 1 (LRG1), is elevated in the atherosclerotic tissue and serum samples from patients with coronary artery disease (CAD). LRG1 stimulated macrophages to proinflammatory M1-like polarization through the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) pathways. The LRG1 knockout mice showed significantly delayed atherogenesis progression and reduced levels of macrophage-related proinflammatory cytokines in a high-fat diet-induced Apoe-/- mouse atherosclerosis model. An anti-LRG1 neutralizing antibody also effectively blocked LRG1-induced macrophage M1-like polarization in vitro and conferred therapeutic benefits to animals with ApoE deficiency-induced atherosclerosis. LRG1 may therefore serve as an additional biomarker for CAD and targeting LRG1 could offer a potential therapeutic strategy for CAD patients by mitigating the proinflammatory response of macrophages.

A novel lncRNA GM47544 modulates triglyceride metabolism by inducing ubiquitination-dependent protein degradation of APOC3

ObjectiveEmerging evidence highlights the pivotal roles of long non-coding RNAs (lncRNAs) in lipid metabolism. Apoprotein C3 (ApoC3) is a well-established therapeutic target for hypertriglyceridemia and exhibits a strong association with cardiovascular disease. However, the exact mechanisms via which the lncRNAs control ApoC3 expression remain unclear. MethodsWe identified a novel long noncoding RNA (lncRNA), GM47544, within the ApoA1/C3/A4/A5 gene cluster. Subsequently, the effect of GM47544 on intracellular triglyceride metabolism was analyzed. The diet-induced mouse models of hyperlipidemia and atherosclerosis were established to explore the effect of GM47544 on dyslipidemia and plaque formation in vivo. The molecular mechanism was explored through RNA sequencing, immunoprecipitation, RNA pull-down assay, and RNA immunoprecipitation. ResultsGM47544 was overexpressed under high-fat stimulation. GM47544 effectively improved hepatic steatosis, reduced blood lipid levels, and alleviated atherosclerosis in vitro and in vivo. Mechanistically, GM47544 directly bound to ApoC3 and facilitated the ubiquitination at lysine 79 in ApoC3, thereby facilitating ApoC3 degradation via the ubiquitin-proteasome pathway. Moreover, we identified AP006216.5 as the human GM47544 transcript, which fulfills a comparable function in human hepatocytes. ConclusionsThe identification of GM47544 as a lncRNA modulator of ApoC3 reveals a novel mechanism of post-translational modification, with significant clinical implications for the treatment of hypertriglyceridemia and atherosclerosis.