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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 αvß3 expressed in atherosclerotic plaques was performed. Pathological assessments verified the imaging results in terms of integrin αvß3 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 αvß3 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.
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Fluorocarburos , Oro , Nanotubos , Péptidos Cíclicos , Técnicas Fotoacústicas , Placa Aterosclerótica , Animales , Placa Aterosclerótica/diagnóstico por imagen , Técnicas Fotoacústicas/métodos , Oro/química , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacocinética , Ratones , Nanotubos/química , Fluorocarburos/química , Integrina alfaVbeta3/metabolismo , Sondas Moleculares/química , Sondas Moleculares/farmacocinética , Ultrasonografía/métodos , Tamaño de la Partícula , Masculino , Ratones Noqueados para ApoE , Modelos Animales de Enfermedad , PentanosRESUMEN
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.
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Apoptosis , Aterosclerosis , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Exosomas , Ratones Endogámicos C57BL , MicroARNs , Músculo Liso Vascular , Miocitos del Músculo Liso , Placa Aterosclerótica , Animales , MicroARNs/metabolismo , MicroARNs/genética , Exosomas/metabolismo , Exosomas/patología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Aterosclerosis/genética , Proliferación Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Miocitos del Músculo Liso/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Músculo Liso Vascular/patología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Masculino , Transducción de Señal , Células Cultivadas , Obesidad/metabolismo , Obesidad/patología , Ratones Noqueados para ApoE , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Endoteliales/efectos de los fármacos , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/genética , Becaplermina/farmacología , Becaplermina/metabolismo , Grasa Intraabdominal/metabolismo , Grasa Intraabdominal/patología , Ratones , HumanosRESUMEN
BACKGROUND: Dyslipidemia increases cardiovascular disease risk, the leading cause of death worldwide. Under time-restricted feeding (TRF), wherein food intake is restricted to a consistent window of <12 hours, weight gain, glucose intolerance, inflammation, dyslipidemia, and hypercholesterolemia are all reduced in mice fed an obesogenic diet. LDLR (low-density lipoprotein receptor) mutations are a major cause of familial hypercholesterolemia and early-onset cardiovascular disease. METHODS: We subjected benchmark preclinical models, mice lacking LDLR-knockout or ApoE knockout to ad libitum feeding of an isocaloric atherogenic diet either ad libitum or 9 hours TRF for up to 13 weeks and assessed disease development, mechanism, and global changes in hepatic gene expression and plasma lipids. In a regression model, a subset of LDLR-knockout mice were ad libitum fed and then subject to TRF. RESULTS: TRF could significantly attenuate weight gain, hypercholesterolemia, and atherosclerosis in mice lacking the LDLR-knockout mice under experimental conditions of both prevention and regression. In LDLR-knockout mice, increased hepatic expression of genes mediating ß-oxidation during fasting is associated with reduced VLDL (very-low-density lipoprotein) secretion and lipid accumulation. Additionally, increased sterol catabolism coupled with fecal loss of cholesterol and bile acids contributes to the atheroprotective effect of TRF. Finally, TRF alone or combined with a cholesterol-free diet can reduce atherosclerosis in LDLR-knockout mice. However, mice lacking ApoE, which is an important protein for hepatic lipoprotein reuptake do not respond to TRF. CONCLUSIONS: In a preclinical animal model, TRF is effective in both the prevention and regression of atherosclerosis in LDLR knockout mice. The results suggest TRF alone or in combination with a low-cholesterol diet can be a lifestyle intervention for reducing cardiovascular disease risk in humans.
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Aterosclerosis , Modelos Animales de Enfermedad , Hígado , Ratones Noqueados para ApoE , Receptores de LDL , Animales , Receptores de LDL/genética , Receptores de LDL/deficiencia , Aterosclerosis/prevención & control , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/etiología , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Factores de Tiempo , Ayuno/sangre , Ratones , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Hipercolesterolemia/complicaciones , Dieta Aterogénica , Aumento de Peso , Ratones Noqueados , Enfermedades de la Aorta/prevención & control , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/metabolismo , Lípidos/sangre , Apolipoproteínas ERESUMEN
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.
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Medicamentos Herbarios Chinos , Transportador de Glucosa de Tipo 1 , Macrófagos , Placa Aterosclerótica , Animales , Placa Aterosclerótica/tratamiento farmacológico , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Ratones , Masculino , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Transportador de Glucosa de Tipo 1/genética , Dieta Alta en Grasa , Ratones Endogámicos C57BL , Fagocitosis/efectos de los fármacos , Humanos , Simulación del Acoplamiento Molecular , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Modelos Animales de Enfermedad , Apoptosis/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Lipoproteínas LDL/metabolismo , Células RAW 264.7 , Ratones Noqueados para ApoE , EferocitosisRESUMEN
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.
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Aterosclerosis , ADN Mitocondrial , Inflamación , Macrófagos , Proteínas de la Membrana , Placa Aterosclerótica , Molécula 1 de Adhesión Celular Vascular , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Animales , Molécula 1 de Adhesión Celular Vascular/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Aterosclerosis/genética , Macrófagos/metabolismo , Humanos , Inflamación/metabolismo , Inflamación/patología , Inflamación/genética , Ratones , Placa Aterosclerótica/patología , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Masculino , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Mitocondrias/patología , Ratones Noqueados para ApoE , Transducción de Señal , Femenino , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismoRESUMEN
BACKGROUND AND AIMS: Janus kinase 2 (JAK2) triggers endothelial pyroptosis and is associated with a multitude of pathological cardiovascular manifestations, including atherosclerosis. However, the associated transcriptional regulatory mechanisms remain unclear. In this study, we investigated a novel transcriptional regulator upstream of JAK2. METHODS: We validated the binding and regulation of Forkhead box C1 (FOXC1) and JAK2 using chromatin immunoprecipitation and luciferase reporter assays. Immunofluorescence was used to detect protein localization in cells and tissues. Immunohistochemistry, hematoxylin-eosin (HE), Masson's trichrome, and Oil Red O staining were used to identify tissue lesions. Transcriptional functions were investigated using in vitro and in vivo coronary artery disease (CAD) atherosclerosis models. RESULTS: The mRNA levels of JAK2 were considerably higher in both the cardiac tissues of mice and the peripheral blood of patients with CAD than in equivalent controls. JAK2 expression increased markedly in the coronary arteries of ApoeKO mice, whereas FOXC1 expression exhibited a decreasing trend. In vitro, FOXC1 bound to the JAK2 promoter region and inversely regulated the expression of JAK2. Mechanistic studies have revealed that the FOXC1-JAK2 pathway regulates pyroptosis and participates in the pathogenesis of human coronary artery endothelial cells (HCAECs). In vivo, the suppression of FOXC1 was confirmed to stimulate the levels of JAK2 and pyroptosis, contributing to the pathological progression of aortic and coronary artery damage. CONCLUSIONS: We established the FOXC1-JAK2 regulatory pathway and verified its reverse-regulatory function in CAD pyroptosis. Our data emphasizes that FOXC1 is critical for the treatment of pyroptosis-induced injury in patients with CAD.
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Enfermedad de la Arteria Coronaria , Vasos Coronarios , Factores de Transcripción Forkhead , Janus Quinasa 2 , Piroptosis , Animales , Janus Quinasa 2/metabolismo , Janus Quinasa 2/genética , Humanos , Vasos Coronarios/patología , Vasos Coronarios/metabolismo , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Enfermedad de la Arteria Coronaria/patología , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/genética , Ratones , Células Endoteliales/enzimología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Masculino , Transducción de Señal , Modelos Animales de Enfermedad , Ratones Noqueados para ApoE , Ratones Endogámicos C57BL , Regiones Promotoras GenéticasRESUMEN
BACKGROUND: HCC-1 (hemofiltrate CC chemokine-1), a CC-type chemokine, exerts function to change intracellular calcium concentration, induce leukocyte, and manipulate enzyme release especially in monocytes. It has been reported that HCC-1 can predict the persistent acute kidney injury or suppress hepatocellular carcinoma by modulating cell cycle and promoting apoptosis; however, the effect of HCC-1 on atherosclerosis is poorly understood. Here, we aimed to clarify the function and mechanism of HCC-1 in atherosclerosis and whether it could serve as a novel biomarker for the diagnosis of atherosclerosis. METHODS: HCC-1 expression in serum, atherosclerotic plaques, and normal arterial tissue from patients with atherosclerosis and control group was assessed by ELISA, immunohistochemistry and confocal microscope, and bioinformatic analysis. The atherosclerotic model of HCC-1 overexpressing and control mice was generated by tail vein injection of adeno-associated virus serotype 9-HCC-1 on an ApoE-/- background. Cell adhesion, polarization, and pyroptosis were evaluated in vitro. The relationship between HCC-1 concentration in serum and atherosclerosis was analyzed in patients with atherosclerosis. RESULTS: HCC-1 expression was positively correlated with the occurrence and stable-unstable switch of atherosclerosis under bioinformatic analysis, which is further supported by the results of increased HCC-1 expression in atherosclerosis patients both in serum and atherosclerotic plaque. adeno-associated virus serotype 9-HCC-1 mice had higher levels of inflammatory factors, increased macrophage accumulation and pyroptotic rate in plaque, and decreased atherosclerotic plaque stability. In vitro, HCC-1 promoted monocyte adhesion and M1 polarization and induced inflammation and pyroptosis both in endothelial cells and macrophages. CONCLUSIONS: HCC-1 expression was increased in patients with atherosclerosis, and HCC-1 overexpression accelerated atherosclerotic burden via an enhancement in monocyte recruitment, M1 polarization, and pyroptosis both in endothelial cells and macrophages. Our findings suggested that HCC-1 may serve as an early biomarker for the diagnosis of atherosclerosis, with the capacity to reflect the degree of stenosis.
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Aterosclerosis , Biomarcadores , Células Endoteliales , Macrófagos , Piroptosis , Humanos , Animales , Aterosclerosis/patología , Aterosclerosis/metabolismo , Aterosclerosis/genética , Aterosclerosis/sangre , Macrófagos/metabolismo , Biomarcadores/sangre , Biomarcadores/metabolismo , Masculino , Persona de Mediana Edad , Femenino , Ratones , Células Endoteliales/metabolismo , Células Endoteliales/patología , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Placa Aterosclerótica , Diagnóstico Precoz , Estudios de Casos y Controles , Ratones Noqueados para ApoE , Anciano , Valor Predictivo de las Pruebas , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Proteínas Reguladoras de la Apoptosis , Receptores DepuradoresRESUMEN
Dysregulation of the hematopoietic niche during hyperlipidemia facilitates pathologic leukocyte production, driving atherogenesis. Although definitive hematopoiesis occurs primarily in the bone marrow, during atherosclerosis this also occurs in the spleen. Cells of the bone marrow niche, particularly endothelial cells, have been studied in atherosclerosis, although little is known about how splenic endothelial cells respond to the atherogenic environment. Here we show unique dysregulated pathways in splenic compared to bone marrow endothelial cells during atherosclerosis, including perturbations of lipid metabolism and endocytic trafficking pathways. As part of this response, we identify the mixed lineage kinase domain-like (MLKL) protein as a repressor of splenic, but not bone marrow, myelopoiesis. Silencing MLKL in splenic endothelial cells results in inefficient endosomal trafficking and lipid accumulation, ultimately promoting the production of myeloid cells that participate in plaque development. These studies identify endocytic trafficking by MLKL as a key mechanism of splenic endothelial cell maintenance, splenic hematopoiesis and, subsequently, atherosclerosis.
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Aterosclerosis , Células Endoteliales , Hiperlipidemias , Proteínas Quinasas , Bazo , Bazo/patología , Bazo/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Quinasas/genética , Células Endoteliales/metabolismo , Células Endoteliales/patología , Animales , Aterosclerosis/patología , Aterosclerosis/metabolismo , Hiperlipidemias/metabolismo , Hiperlipidemias/patología , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Masculino , Mielopoyesis , Humanos , Células Cultivadas , Metabolismo de los Lípidos , Ratones , Placa Aterosclerótica/patología , Placa Aterosclerótica/metabolismo , Ratones Noqueados para ApoE , Endocitosis/fisiología , Endosomas/metabolismo , Nicho de Células Madre/fisiologíaRESUMEN
BACKGROUND AND AIMS: Inflammatory cells within atherosclerotic lesions secrete proteolytic enzymes that contribute to lesion progression and destabilization, increasing the risk for an acute cardiovascular event. Elastase is a serine protease, secreted by macrophages and neutrophils, that may contribute to the development of unstable plaque. We previously reported interaction of endogenous protease-inhibitor proteins with high-density lipoprotein (HDL), including alpha-1-antitrypsin, an inhibitor of elastase. These findings support a potential role for HDL as a modulator of protease activity. In this study, we test the hypothesis that enhancement of HDL-associated elastase inhibitor activity is protective against atherosclerotic lesion progression. METHODS: We designed an HDL-targeting protease inhibitor (HTPI) that binds to HDL and confers elastase inhibitor activity. Lipoprotein binding and the impact of HTPI on atherosclerosis were examined using mouse models. Histology and immunofluorescence staining of aortic root sections were used to examine the impact of HTPI on lesion morphology and inflammatory features. RESULTS: HTPI is a small (1.6 kDa) peptide with an elastase inhibitor domain, a soluble linker, and an HDL-targeting domain. When incubated with human plasma ex vivo, HTPI predominantly binds to HDL. Intravenous administration of HTPI to mice resulted in its binding to plasma HDL and increased elastase inhibitor activity on isolated HDL. Accumulation of HTPI within plaque was observed after administration to Apoe-/- mice. To examine the effect of HTPI treatment on atherosclerosis, prevention and progression studies were performed using Ldlr-/- mice fed Western diet. In both study designs, HTPI-treated mice had reduced lipid deposition in plaque. CONCLUSIONS: These data support the hypothesis that HDL-associated anti-elastase activity can improve the atheroprotective potential of HDL and highlight the potential utility of HDL enrichment with anti-protease activity as an approach for stabilization of atherosclerotic lesions.
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Aterosclerosis , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Lipoproteínas HDL , Animales , Aterosclerosis/patología , Aterosclerosis/prevención & control , Aterosclerosis/enzimología , Aterosclerosis/metabolismo , Aterosclerosis/tratamiento farmacológico , Lipoproteínas HDL/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones , Ratones Noqueados para ApoE , Placa Aterosclerótica , Masculino , Elastasa Pancreática/metabolismo , Aorta/patología , Aorta/efectos de los fármacos , Aorta/enzimología , Aorta/metabolismo , Enfermedades de la Aorta/prevención & control , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/enzimología , Enfermedades de la Aorta/metabolismo , Inhibidores de Proteasas/farmacología , alfa 1-Antitripsina/farmacología , alfa 1-Antitripsina/metabolismoRESUMEN
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)-âº. Thus, we also explored whether inhibiting TNF-⺠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.
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Apolipoproteínas E , Aterosclerosis , Péptido Relacionado con Gen de Calcitonina , Ratones Noqueados , Placa Aterosclerótica , Animales , Aterosclerosis/metabolismo , Aterosclerosis/genética , Aterosclerosis/patología , Péptido Relacionado con Gen de Calcitonina/metabolismo , Ratones , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Placa Aterosclerótica/patología , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/genética , Dieta Alta en Grasa/efectos adversos , Factor de Necrosis Tumoral alfa/metabolismo , Masculino , Ratones Noqueados para ApoE , Modelos Animales de Enfermedad , Humanos , Anticuerpos Monoclonales Humanizados/farmacología , Etanercept/farmacología , Ratones Endogámicos C57BL , Movimiento Celular/efectos de los fármacos , Aorta/metabolismo , Aorta/patología , Aorta/efectos de los fármacosRESUMEN
Human aortic vascular smooth muscle cells (HA-VSMCs) play vital roles in the pathogenesis of vascular diseases, including Atherosclerosis (AS). Circular RNAs (circRNAs) have been reported to regulate the biological functions of HA-VSMCs. Therefore, this study aimed to explore the role and mechanism of hsa_circRNA_102353 (circ_0007765) in platelet-derived growth factor-BB (PDGF-BB)-induced HA-VSMCs. Circ_0007765, microRNA-654-3p (miR-654-3p), and Fibroblast Growth Factor Receptor Substrate 2 (FRS2) expression were measured using real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferative ability, invasion, and migration were detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), Transwell, and wound healing assays. CyclinD1, MMP2, and FRS2 protein levels were assessed using a Western blot assay. Binding between miR-654-3p and circ_0007765 or FRS2 was predicted by Circinteractome or TargetScan, and verified using dual-luciferase reporter and RNA pull-down assays. PDGF-BB induced HA-VSMC proliferation, invasion, and migration. Circ_0007765 and FRS2 expression levels were increased in PDGF-BB-treated HA-VSMCs, and the miR-654-3p level was reduced. Moreover, circ_0007765 absence hindered PDGF-BB-induced HA-VSMC proliferation, invasion, and migration in vitro. At the molecular level, circ_0007765 increased FRS2 expression by acting as a sponge for miR-654-3p. Our findings revealed that circ_0007765 boosted PDGF-BB-induced HA-VSMC proliferation and migration through elevating FRS2 expression via adsorbing miR-654-3p, providing a feasible therapeutic strategy for AS.
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Proteínas Adaptadoras Transductoras de Señales , Aterosclerosis , Becaplermina , Movimiento Celular , Proliferación Celular , Proteínas de la Membrana , MicroARNs , Músculo Liso Vascular , Miocitos del Músculo Liso , ARN Circular , Transducción de Señal , Humanos , Músculo Liso Vascular/patología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , ARN Circular/metabolismo , ARN Circular/genética , Becaplermina/farmacología , Movimiento Celular/efectos de los fármacos , Miocitos del Músculo Liso/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , MicroARNs/metabolismo , MicroARNs/genética , Aterosclerosis/patología , Aterosclerosis/metabolismo , Aterosclerosis/genética , Células Cultivadas , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Aorta/patología , Aorta/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/genética , Regulación de la Expresión Génica , Ratones Noqueados para ApoE , AnimalesRESUMEN
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.
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Aterosclerosis , Glicoproteínas , Macrófagos , Animales , Aterosclerosis/patología , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/inmunología , Macrófagos/metabolismo , Macrófagos/inmunología , Ratones , Humanos , Glicoproteínas/metabolismo , Glicoproteínas/genética , Ratones Noqueados , Masculino , Apolipoproteínas E/genética , Apolipoproteínas E/deficiencia , Apolipoproteínas E/metabolismo , Modelos Animales de Enfermedad , Citocinas/metabolismo , Dieta Alta en Grasa/efectos adversos , Ratones Endogámicos C57BL , Enfermedad de la Arteria Coronaria/patología , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/inmunología , Femenino , Ratones Noqueados para ApoE , Activación de MacrófagosRESUMEN
The onset and progression of atherosclerosis are closely linked to the involvement of macrophages. While the contribution of NLRP3 inflammasome activation to the creation of a local highly inflammatory microenvironment is well recognized, the precise triggers remain unclear. In this study, we aimed to investigate the regulatory mechanism of NLRP3 inflammasome activation in response to hypoxia-induced glycolysis involving PFKFB3 in the development of atherosclerosis. To develop an atherosclerosis model, we selected ApoE knockout mice treated with a high-fat western diet. We then quantified the expression of HIF-1α, PFKFB3, and NLRP3. In addition, we administered the PFKFB3 inhibitor PFK158 during atherosclerosis modeling. The glycolytic activity was subsequently determined through 18F-FDG micro-PET/CT, ex vivo glucose uptake, and ECAR analysis. Furthermore, we employed lipopolysaccharide (LPS) and TNF-α to induce the differentiation of bone marrow-derived macrophages (BMDMs) into M1-like phenotypes under both hypoxic and normoxic conditions. Our histological analyses revealed the accumulation of PFKFB3 in human atherosclerotic plaques, demonstrating colocalization with NLRP3 expression and macrophages. Treatment with PFK158 reduced glycolytic activity and NLRP3 inflammasome activation, thereby mitigating the occurrence of atherosclerosis. Mechanistically, hypoxia promoted glycolytic reprogramming and NLRP3 inflammasome activation in BMDMs. Subsequent blocking of either HIF-1α or PFKFB3 downregulated the NLRP3/Caspase-1/IL-1ß pathway in hypoxic BMDMs. Our study demonstrated that the HIF-1α/PFKFB3/NLRP3 axis serves as a crucial mechanism for macrophage inflammation activation in the emergence of atherosclerosis. The therapeutic potential of PFKFB3 inhibition may represent a promising strategy for atheroprotection.
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Aterosclerosis , Glucólisis , Inflamasomas , Macrófagos , Proteína con Dominio Pirina 3 de la Familia NLR , Fosfofructoquinasa-2 , Animales , Fosfofructoquinasa-2/metabolismo , Fosfofructoquinasa-2/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Ratones , Macrófagos/metabolismo , Inflamasomas/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Masculino , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Hipoxia/metabolismo , Ratones NoqueadosRESUMEN
Atherosclerosis (AS) is the main pathological basis of cardiovascular diseases such as coronary heart disease. Black phosphorus quantum dots (BPQDs) are a novel nanomaterial with good optical properties and biocompatibility, which was applied in the treatment of AS in mice, with good results shown in our previous study. In this study, BPQDs were injected into high-fat diet-fed apolipoprotein E knockout mice as a preventive drug for 12 weeks. Simvastatin, a classic preventive drug for AS, was used as a control to verify the preventive effect of BPQDs. The results showed that after preventive treatment with BPQDs, the plaque area in mice was significantly reduced, the vascular elasticity was increased, and serum lipid levels were significantly lower than those in the model group. To explore the mechanism, macrophages were induced to become foam cells using oxidized low-density lipoprotein. We found that BPQDs treatment could increase cell autophagy, thereby regulating intracellular lipid metabolism. Taken together, these data revealed that BPQDs may serve as a functional drug in preventing the development of AS.
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Aterosclerosis , Dieta Alta en Grasa , Fósforo , Puntos Cuánticos , Animales , Dieta Alta en Grasa/efectos adversos , Aterosclerosis/prevención & control , Ratones , Fósforo/sangre , Ratones Noqueados , Apolipoproteínas E/genética , Masculino , Autofagia/efectos de los fármacos , Ratones Noqueados para ApoE , Metabolismo de los Lípidos/efectos de los fármacos , Modelos Animales de Enfermedad , Placa Aterosclerótica/prevención & control , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/sangre , Simvastatina/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Células Espumosas/efectos de los fármacos , Células Espumosas/metabolismoRESUMEN
BACKGROUND: Atherosclerosis is highly prevalent in people with chronic kidney disease (CKD), including those receiving peritoneal dialysis (PD). Although it is lifesaving, PD induces profound systemic inflammation, which may aggravate atherosclerosis. Therefore, the hypothesis is that this PD-induced inflammation aggravates atherosclerosis via immune cell activation. METHODS AND RESULTS: ApoE-/- mice were subjected to a 5/6 nephrectomy to induce CKD. Three weeks later, mice were fed a high-cholesterol diet. Half of the nephrectomized mice then received daily peritoneal infusions of 3.86% Physioneal for 67 further days (CKD+PD) until the end of the experiment, and were compared with mice without CKD. Sham operated and PD-only mice were additional controls. CKD+PD mice displayed more severe atherosclerotic disease than control mice. Plaque area increased, and plaques were more advanced with a vulnerable phenotype typified by decreased collagen content and decreased fibrous cap thickness. Increased CD3+ T-cell numbers were present in plaques and perivascular adipose tissue of CKD and CKD+PD mice. Plaques of CKD+PD mice contained more iNOS+ immune cells. Spleens of CKD+PD mice showed more CD4+ central memory, terminally differentiated type 1 T-helper (Th1), Th17, and CX3C motif chemokine receptor 1+ (CX3CR1) CD4+ T-cells with less regulatory and effector T-cells. CONCLUSIONS: PD-fluid exposure in uremic mice potentiates systemic and vascular T-cell-driven inflammation and aggravates atherosclerosis. PD polarized CD4+ T-cells toward an inflammatory Th1/Th17 phenotype, and increased CX3CR1+ CD4+ T-cells, which are associated with vascular homing in CKD-associated atherosclerosis. Targeting CD4+ T-cell activation and CX3CR1+ polarization has the potential to attenuate atherosclerosis in PD patients.
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Aterosclerosis , Modelos Animales de Enfermedad , Diálisis Peritoneal , Insuficiencia Renal Crónica , Uremia , Animales , Aterosclerosis/patología , Aterosclerosis/etiología , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Aterosclerosis/genética , Uremia/inmunología , Uremia/metabolismo , Diálisis Peritoneal/efectos adversos , Insuficiencia Renal Crónica/inmunología , Insuficiencia Renal Crónica/metabolismo , Ratones Noqueados para ApoE , Ratones , Placa Aterosclerótica , Masculino , Ratones Endogámicos C57BL , Apolipoproteínas E/genética , Apolipoproteínas E/deficiencia , NefrectomíaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Qinghao-Biejia herb pair (QB) is the core herb pair of "Jieduquyuziyin prescription" and is one of the commonly used herb pairs for the clinical treatment of systemic lupus erythematosus (SLE). Previous studies have shown that QB reduces the expression of inflammatory cytokines like IL-6 and TNF-α in the serum and kidney of MRL/lpr mice. Additionally, it inhibits the expression of TLR4 and MyD88 in the kidney and aorta and reduces the deposition of renal complement C3 and aortic plaque after treatment. These findings suggest that QB has a preventive and therapeutic effect on lupus rats. AIM OF THE STUDY: This study sought to investigate the mechanisms underlying the anti-SLE combined with atherosclerosis activity of the Qinghao-Biejia herb pair. MATERIALS AND METHODS: Drug targets for QB were identified using the HERB database, while targets associated with SLE and atherosclerosis were retrieved from the GeneCards database. The intersection of these drug and disease targets was then analyzed using a protein-protein interaction (PPI) network with GO and KEGG pathway enrichment analysis. In vivo, apolipoprotein E-deficient (ApoE-/-) mice were induced to develop SLE-AS by intraperitoneal injection of pristane and continued feeding of a high-fat diet. The changes in relevant indexes were observed after 12 weeks of gavage treatment with hydroxychloroquine, QB, Q (Qinghao alone), and B (Biejia alone). Bone marrow-derived macrophages from ApoE-/- mice and Raw 264.7 macrophages were used to explore the mechanisms of QB treatment. RESULTS: The levels of inflammatory cytokines in serum and pathological liver changes in mice were improved to varying degrees in the treatment groups. Additionally, there was a reduction in aortic atheromatous plaque formation and some improvement in cholesterol efflux. Furthermore, QB suppressed the expression of inflammatory cytokines in M1 macrophages, suggesting a role in regulating macrophage polarization. CONCLUSION: QB demonstrates clear efficacy for treating SLE-AS, and its therapeutic mechanism may involve the regulation of macrophage phenotypes by promoting cholesterol efflux.
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Transportador 1 de Casete de Unión a ATP , Aterosclerosis , Colesterol , Medicamentos Herbarios Chinos , Lupus Eritematoso Sistémico , Macrófagos , Animales , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Aterosclerosis/tratamiento farmacológico , Ratones , Lupus Eritematoso Sistémico/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Transportador 1 de Casete de Unión a ATP/metabolismo , Colesterol/sangre , Colesterol/metabolismo , Células RAW 264.7 , Ratones Endogámicos C57BL , Femenino , Ratones Endogámicos MRL lpr , Ratones Noqueados para ApoERESUMEN
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-6Chigh 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.
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Aterosclerosis , Macrófagos , Mitocondrias , Necrosis , Peptidil-Prolil Isomerasa F , Placa Aterosclerótica , Animales , Peptidil-Prolil Isomerasa F/metabolismo , Peptidil-Prolil Isomerasa F/genética , Macrófagos/metabolismo , Aterosclerosis/patología , Aterosclerosis/metabolismo , Aterosclerosis/genética , Ratones , Mitocondrias/metabolismo , Mitocondrias/patología , Células RAW 264.7 , Modelos Animales de Enfermedad , Apoptosis , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Necroptosis , Masculino , Ratones Noqueados , Apolipoproteínas E/genética , Apolipoproteínas E/deficiencia , Ciclofilinas/metabolismo , Ciclofilinas/genética , Ciclofilinas/deficiencia , Dieta Alta en Grasa , Interleucina-1beta/metabolismo , Antígenos LyRESUMEN
BACKGROUND AND AIMS: Diabetes is one of the major causes of cardiovascular disease (CVD). As high as 29 % of patients with diabetes develop atherosclerosis. Vascular Smooth Muscle Cells (VSMCs) are a key mediator in the pathogenesis of atherosclerosis, generating pro-inflammatory and proliferative characteristics in atherosclerotic lesions. METHODS: We used human atherosclerotic samples, developed diabetes-induced atherosclerotic mice, and generated loss of function and gain of function in Klotho human aortic smooth muscle cells to investigate the function of Klotho in atherosclerosis. RESULTS: We found that Klotho expression is decreased in smooth muscle actin-positive cells in patients with diabetes and atherosclerosis. Consistent with human data, we found that Apoe knockout mice with streptozotocin-induced diabetes fed on a high-fat diet showed decreased expression of Klotho in SMCs. Additionally, these mice showed increased expression of TGF-ß, MMP9, phosphorylation of ERK and Akt. Further, we utilized primary Human Aortic Smooth Muscle Cells (HASMCs) with d-glucose under dose-response and in time-dependent conditions to study the role of Klotho in these cells. Klotho gain of function and loss of function studies showed that Klotho inversely regulated the expression of atherosclerotic markers TGF-ß, MMP2, MMP9, and Fractalkine. Further, High Glucose (HG) induced Akt, and ERK1/2 phosphorylation were enhanced or mitigated by endogenous Klotho deficiency or its overexpression respectively. PI3K/Akt and MAPK/ERK inhibition partially abolished the HG-induced upregulation of TGF-ß, MMP2, MMP9, and Fractalkine. Additionally, Klotho knockdown increased the proliferation of HASMCs and enhanced α-SMA and TGF-ß expression. CONCLUSIONS: Taken together, these results indicate that local vascular Klotho is involved in diabetes-induced atherosclerosis, which is via PI3K/Akt and ERK1/2-dependent signaling pathways.
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Aterosclerosis , Diabetes Mellitus Experimental , Glucuronidasa , Proteínas Klotho , Ratones Noqueados para ApoE , Músculo Liso Vascular , Miocitos del Músculo Liso , Proteínas Klotho/metabolismo , Animales , Aterosclerosis/metabolismo , Aterosclerosis/patología , Aterosclerosis/genética , Glucuronidasa/metabolismo , Glucuronidasa/genética , Humanos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/complicaciones , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Masculino , Transducción de Señal , Células Cultivadas , Aorta/patología , Aorta/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/enzimología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proliferación CelularRESUMEN
BACKGROUND: Atherosclerosis is a long-lasting inflammatory condition affecting the walls of arteries, marked by the buildup of fats, plaque formation, and vascular remodeling. Recent findings highlight the significance of cholesterol removal pathways in influencing atherosclerosis, yet the connection between cholesterol removal and regulation of macrophage inflammation remains poorly understood. RBAP could serve as an anti-inflammatory agent; however, its role in atherosclerosis and the mechanism behind it are still not well understood. PURPOSE: The objective of this research is to explore how RBAP impacts cholesterol efflux, which is a considerable element in the advancement of atherosclerosis. METHODS: An atherosclerosis mouse model was established by using an ApoE KO strain mouse on a high-fat diet (HFD) to assess the effects of RBAP, conducted either orally or through injection. Additionally, in vitro experiments were conducted where the induction of THP-1 cells was conducted for the differentiation towards macrophages, and along with mouse RAW264.7 cells, were challenged with ox-LDL to evaluate the impact of RBAP. RESULTS: In this study, RBAP was found to reduce the production and downregulate TNF-α, IL-1ß, and IL-6 levels and inhibited the activation of the TLR4/MyD88/NF-κB signaling in atherosclerosis model mice, as well as in ox-LDL-challenged THP-1 cells and mouse RAW264.7 macrophages. RBAP's effectiveness also improved the enhancement of reverse cholesterol transport (RCT) and cholesterol removal to HDL and apoA1 by increasing the activity of genes related to cholesterol removal PPARγ/LXRα/ABCA1/ABCG1, both in ApoE-/- mice and in THP-1 cells and mouse RAW264.7 macrophages. Notably, RBAP exerted similar effects on atherosclerosis model mice and macrophages to those of TAK-242, an inhibitor of the TLR4 signaling. When RBAP and TAK-242 were applied simultaneously, the improvement was not enhanced compared with either RBAP or TAK-242 treatment alone. CONCLUSION: These findings suggest that RBAP, as a TLR4 inhibitor, has anti-atherosclerotic effects by improving inflammation and promoting cholesterol effection, indicating its therapeutic potential in intervening atherosclerosis.
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Aterosclerosis , Diferenciación Celular , Colesterol , Células Espumosas , Macrófagos , Oryza , Receptor Toll-Like 4 , Animales , Aterosclerosis/tratamiento farmacológico , Ratones , Colesterol/metabolismo , Células Espumosas/efectos de los fármacos , Células Espumosas/metabolismo , Células RAW 264.7 , Diferenciación Celular/efectos de los fármacos , Humanos , Receptor Toll-Like 4/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Modelos Animales de Enfermedad , Células THP-1 , Masculino , Dieta Alta en Grasa , Transportador 1 de Casete de Unión a ATP/metabolismo , Lipoproteínas LDL/metabolismo , Ratones Endogámicos C57BL , Péptidos/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/metabolismo , Ratones Noqueados para ApoE , FN-kappa B/metabolismo , Apolipoproteínas E , Antiinflamatorios/farmacologíaRESUMEN
BACKGROUND: Atherosclerosis (AS) is the main pathological basis for the development of cardiovascular diseases. Vascular inflammation is an important factor in the formation of AS, and macrophage pyroptosis plays a key role in AS due to its unique inflammatory response. Guizhitongluo Tablet (GZTLT) has shown clinically effective in treating patients with AS, but its mechanism is elusive. PURPOSE: This study was to determine the effects of GZTLT on atherosclerotic vascular inflammation and pyroptosis and to understand its underlying mechanism. MATERIALS AND METHODS: The active constituents of GZTLT were analysed by means of UPLC-HRMS. In vivo experiments were performed using ApoE-/- mice fed a high fat diet for 8 weeks, followed by treatment with varying concentrations of GZTLT orally by gavage and GsMTx4 (GS) intraperitoneally and followed for another 8 weeks. Oil red O, Haematoxylin-eosin (HE) and Masson staining were employed to examine the lipid content, plaque size, and collagen fibre content of the mouse aorta. Immunofluorescence staining was utilised to identify macrophage infiltration, as well as the expression of Piezo1 and NLRP3 proteins in aortic plaques. The levels of aortic inflammatory factors were determined using RT-PCR and ELISA. In vitro, foam cell formation in bone marrow-derived macrophages (BMDMs) was observed using Oil Red O staining. Intracellular Ca2+ measurements were performed to detect the calcium influx in BMDMs, and the expression of NLRP3 and its related proteins were detected by Western blot. RESULTS: The UPLC-HRMS analysis revealed 31 major components of GZTLT. Our data showed that GZTLT inhibited aortic plaque formation in mice and increased plaque collagen fibre content to stabilise plaques. In addition, GZTLT could restrain the expression of serum lipid levels and suppress macrophage foam cell formation. Further studies found that GZTLT inhibited macrophage infiltration in aortic plaques and suppressed the expression of inflammatory factors. It is noteworthy that GZTLT can restrain Piezo1 expression and reduce Ca2+ influx in BMDMs. Additionally, we found that GZTLT could regulate NLRP3 activation and pyroptosis by inhibiting Piezo1. CONCLUSION: The present study suggests that GZTLT inhibits vascular inflammation and macrophage pyroptosis through the Piezo1/NLRP3 signaling pathway, thereby delaying AS development. Our finding provides a potential target for AS treatment and drug discovery.