RESUMEN
BACKGROUND: Acute coronary syndrome (ACS) involves plaque-related thrombosis, causing primary ischemic cardiomyopathy or lethal arrhythmia. We previously demonstrated a unique immune landscape of myeloid cells in the culprit plaques causing ACS by using single-cell RNA sequencing. Here, we aimed to characterize T cells in a single-cell level, assess clonal expansion of T cells, and find a therapeutic target to prevent ACS. METHODS: We obtained the culprit lesion plaques from 4 patients with chronic coronary syndrome (chronic coronary syndrome plaques) and the culprit lesion plaques from 3 patients with ACS (ACS plaques) who were candidates for percutaneous coronary intervention with directional coronary atherectomy. Live CD45+ immune cells were sorted from each pooled plaque samples and applied to the 10× platform for single-cell RNA sequencing analysis. We also extracted RNA from other 3 ACS plaque samples and conducted unbiased TCR (T-cell receptor) repertoire analysis. RESULTS: CD4+ T cells were divided into 5 distinct clusters: effector, naive, cytotoxic, CCR7+ (C-C chemokine receptor type 7) central memory, and FOXP3 (forkhead box P3)+ regulatory CD4+ T cells. The proportion of central memory CD4+ T cells was higher in the ACS plaques. Correspondingly, dendritic cells also tended to express more HLAs (human leukocyte antigens) and costimulatory molecules in the ACS plaques. The velocity analysis suggested the differentiation flow from central memory CD4+ T cells into effector CD4+ T cells and that from naive CD4+ T cells into central memory CD4+ T cells in the ACS plaques, which were not observed in the chronic coronary syndrome plaques. The bulk repertoire analysis revealed clonal expansion of TCRs in each patient with ACS and suggested that several peptides in the ACS plaques work as antigens and induced clonal expansion of CD4+ T cells. CONCLUSIONS: For the first time, we revealed single cell-level characteristics of CD4+ T cells in patients with ACS. CD4+ T cells could be therapeutic targets of ACS. REGISTRATION: URL: https://upload.umin.ac.jp/cgi-open-bin/icdr_e/ctr_view.cgi?recptno=R000046521; Unique identifier: UMIN000040747.
Asunto(s)
Síndrome Coronario Agudo , Linfocitos T CD4-Positivos , Placa Aterosclerótica , Análisis de la Célula Individual , Humanos , Síndrome Coronario Agudo/inmunología , Síndrome Coronario Agudo/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Masculino , Persona de Mediana Edad , Femenino , Anciano , RNA-Seq , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Vasos Coronarios/inmunología , Vasos Coronarios/patología , Análisis de Secuencia de ARN , Enfermedad de la Arteria Coronaria/inmunología , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/patología , FenotipoRESUMEN
In the brain, neurons, glial cells, vascular endothelial cells (ECs), and mural cells form a functional structure referred to as the neurovascular unit (NVU). The functions of the NVU become impaired with aging. To gain insight into the mechanism underlying the aging-related changes in the NVU, we characterized in the present study the gliovascular interface in transgenic mice expressing a dominant-negative form of the telomeric repeat-binding factor 2 (TERF2) specifically in ECs using the Tie2 promoter. In these transgenic mice, senescence occurred in the cerebral ECs and was accompanied by upregulation of the mRNAs of proinflammatory cell adhesion molecules and cytokines. It is noteworthy that in the deep layers of the cerebral cortex, astrocytes exhibited an increase in the signals for S100ß as well as a decrease in the polarization of the water channel aquaporin-4 (AQP4) to the perivascular endfeet of the astrocytes. Mechanistically, the perivascular localization of dystroglycan and its ligand, laminin α2, was decreased, and their localization correlated well with the perivascular localization of AQP4, which supports the notion that their interaction regulates the perivascular localization of AQP4. The diminished perivascular localization of laminin α2 may be attributed to its proteolytic degradation by the matrix metalloproteinase-2 released by senescent ECs. Pericyte coverage was increased and negatively correlated with the decrease in the perivascular localization of AQP4. We propose that aging-related changes in ECs induce a mild morphological alteration of astrocytes and affect the localization of AQP4 at the gliovascular interface.
Asunto(s)
Senescencia Celular , Células Endoteliales , Laminina , Metaloproteinasa 2 de la Matriz , Neuroglía , Animales , Ratones , Acuaporina 4/genética , Acuaporina 4/metabolismo , Astrocitos/metabolismo , Células Endoteliales/metabolismo , Laminina/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones Transgénicos , Neuroglía/metabolismoRESUMEN
OBJECTIVE: Chondroitin sulfate proteoglycans are the primary constituents of the macrophage glycosaminoglycan and extracellular microenvironment. To examine their potential role in atherogenesis, we investigated the biological importance of one of the chondroitin sulfate glycosaminoglycan biosynthesis gene, ChGn-2 (chondroitin sulfate N-acetylgalactosaminyltransferase-2), in macrophage foam cell formation. Approach and Results: ChGn-2-deficient mice showed decreased and shortened glycosaminoglycans. ChGn-2-/-/LDLr-/- (low-density lipoprotein receptor) mice generated less atherosclerotic plaque after being fed with Western diet despite exhibiting a metabolic phenotype similar to that of the ChGn-2+/+/LDLr-/- littermates. We demonstrated that in macrophages, ChGn-2 expression was upregulated in the presence of oxLDL (oxidized LDL), and glycosaminoglycan was substantially increased. Foam cell formation was significantly altered by ChGn-2 in both mouse peritoneal macrophages and the RAW264.7 macrophage cell line. Mechanistically, ChGn-2 enhanced oxLDL binding on the cell surface, and as a consequence, CD36-an important macrophage membrane scavenger receptor-was differentially regulated. CONCLUSIONS: ChGn-2 alteration on macrophages conceivably influences LDL accumulation and subsequently accelerates plaque formation. These results collectively suggest that ChGn-2 is a novel therapeutic target amenable to clinical translation in the future. Graphic Abstract: A graphic abstract is available for this article.
Asunto(s)
Aterosclerosis/metabolismo , Células Espumosas/metabolismo , Glicosaminoglicanos/metabolismo , N-Acetilgalactosaminiltransferasas/metabolismo , Animales , Aterosclerosis/etiología , Aterosclerosis/patología , Modelos Animales de Enfermedad , Femenino , Células Espumosas/patología , Glicosaminoglicanos/química , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , N-Acetilgalactosaminiltransferasas/deficiencia , N-Acetilgalactosaminiltransferasas/genética , Placa Aterosclerótica/etiología , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patología , Células RAW 264.7 , Regulación hacia ArribaRESUMEN
Here, we developed a novel in vitro co-culture model, in which process-bearing astrocytes and isolated cerebral microvessels from mice were co-cultured. Astrocytes formed contacts with microvessels from both adult and neonatal mice. However, concentrated localization of the immunofluorescence signal for aquaporin-4 (AQP4) at contact sites between perivascular endfoot processes and blood vessels was only detected with neonatal mouse microvessels. Contact between astrocytic processes and microvessels was retained, whereas concentrated localization of AQP4 signal at contact sites was lost, by knockdown of dystroglycan or α-syntrophin, reflecting polarized localization of AQP4 at perivascular regions in the brain. Further, using our in vitro co-culture model, we found that astrocytes predominantly extend processes to pericytes located at the abluminal surface of microvessels, providing additional evidence that this model is representative of the in vivo situation. Altogether, we have developed a novel in vitro co-culture model that can reproduce aspects of the in vivo situation and is useful for assessing contact formation between astrocytes and blood vessels.
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Astrocitos/fisiología , Encéfalo/fisiología , Microvasos/fisiología , Animales , Acuaporina 4/metabolismo , Astrocitos/metabolismo , Encéfalo/metabolismo , Proteínas de Unión al Calcio/metabolismo , Estructuras de la Membrana Celular/metabolismo , Estructuras de la Membrana Celular/fisiología , Técnicas de Cocultivo/métodos , Distroglicanos/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Microvasos/metabolismo , Proteínas Musculares/metabolismo , Pericitos/metabolismo , Pericitos/fisiologíaRESUMEN
BACKGROUND: It is increasingly recognized that gut microbiota play a pivotal role in the development of atherosclerotic cardiovascular disease. Previously, we have reported that the abundance of genus Bacteroides is lower in patients with coronary artery disease (CAD) than in patients without CAD with coronary risk factors or in healthy volunteers. However, it remains unclear which and how specific gut bacteria contribute to the progression of atherosclerosis. METHODS: We recruited patients with CAD patients and controls without CAD with coronary risk factors. We then compared gut microbial composition using 16S ribosomal RNA gene sequencing in fecal samples to detect species with differential abundance between 2 groups. Subsequently, we used atherosclerosis-prone mice to study the mechanisms underlying the relationship between such species and atherosclerosis. RESULTS: Human fecal 16S ribosomal RNA gene sequencing revealed a significantly lower abundance of Bacteroides vulgatus and Bacteroides dorei in patients with CAD. This significant differential abundance was confirmed by quantitative polymerase chain reaction. Gavage with live B. vulgatus and B. dorei attenuated atherosclerotic lesion formation in atherosclerosis-prone mice, markedly ameliorating endotoxemia followed by decreasing gut microbial lipopolysaccharide production, effectively suppressing proinflammatory immune responses. Furthermore, fecal lipopolysaccharide levels in patients with CAD were significantly higher and negatively correlated with the abundance of B. vulgatus and B. dorei. CONCLUSIONS: Our translational research findings identify a previously unknown link between specific gut bacteria and atherosclerosis. Treatment with live B. vulgatus and B. dorei may help prevent CAD. CLINICAL TRIAL REGISTRATION: URL: https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000018051 . Unique identifier: UMIN000015703.
Asunto(s)
Aterosclerosis/patología , Bacteroides/aislamiento & purificación , Microbioma Gastrointestinal , Lipopolisacáridos/sangre , Anciano , Animales , Aterosclerosis/epidemiología , Aterosclerosis/inmunología , Aterosclerosis/veterinaria , Bacteroides/genética , Heces/microbiología , Femenino , Humanos , Inmunidad Mucosa , Intestinos/inmunología , Lipopolisacáridos/análisis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Factores de Riesgo , Análisis de Secuencia de ARN , Uniones Estrechas/metabolismo , Uniones Estrechas/microbiologíaRESUMEN
Nonalcoholic steatohepatitis (NASH) progresses from nonalcoholic fatty liver disease (NAFLD); however, efficacious drugs for NASH treatment are lacking. Sodium alginate (SA), a soluble dietary fiber extracted from brown algae, could protect the small intestine from enterobacterial invasion. NASH pathogenesis has been suggested to be associated with enterobacterial invasion, so we examined the effect of SA on methionine- and choline-deficient (MCD) diet-induced steatohepatitis in mice (the most widely-used model of NASH). The mice (n = 31) were divided into three groups (mice fed with regular chow, MCD diet, and MCD diet premixed with 5% SA) for 4 and 8 weeks. The MCD diet increased lipid accumulation and inflammation in the liver, the NAFLD Activity Score and hepatic mRNA expression of tumor necrosis factor-ï¡ and collagen 1ï¡1, and induced macrophage infiltration. Villus shortening, disruption of zonula occludens-1 localization and depletion of mucus production were observed in the small intestine of the MCD-group mice. SA administration improved lipid accumulation and inflammation in the liver, and impaired barrier function in the small intestine. Collectively, these results suggest that SA is useful for NASH treatment because it can prevent hepatic inflammation and fatty degeneration by maintaining intestinal barrier function.
Asunto(s)
Alginatos/farmacología , Hígado Graso/tratamiento farmacológico , Metionina/deficiencia , Animales , Deficiencia de Colina/tratamiento farmacológico , Deficiencia de Colina/metabolismo , Deficiencia de Colina/patología , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Modelos Animales de Enfermedad , Hígado Graso/etiología , Hígado Graso/metabolismo , Hígado Graso/patología , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Intestino Delgado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
In the central nervous system, astrocytes extend endfoot processes to ensheath synapses and microvessels. However, the mechanisms underlying this astrocytic process extension remain unclear. A limitation of the use of 2D cultured astrocytes for such studies is that they display a flat, epithelioid morphology, with no or very few processes, which is markedly different from the stellate morphology observed in vivo. In this study, we obtained 2D cultured astrocytes with a rich complexity of processes using differentiation of neurospheres in vitro. Using these process-bearing astrocytes, we showed that laminin, an extracellular matrix molecule abundant in perivascular sites, efficiently induced process formation and branching. Specifically, the numbers of the first- and second-order branch processes and the maximal process length of astrocytes were increased when cultured on laminin, compared with when they were cultured on poly-L-ornithine or type IV collagen. Knockdown of dystroglycan or α-syntrophin, constituent proteins of the dystrophin-glycoprotein complex that provides a link between laminin and the cytoskeleton, using small interference RNAs inhibited astrocyte process formation and branching, and down-regulated expression of the water channel aquaporin-4 (AQP4). Direct knockdown and a specific inhibitor of AQP4 also inhibited, whereas over-expression of AQP4 enhanced astrocyte process formation and branching. Knockdown of AQP4 decreased phosphorylation of focal adhesion kinase (FAK) that is critically implicated in actin remodeling. Collectively, these results indicate that the laminin-dystroglycan-α-syntrophin-AQP4 axis is important for process formation and branching of 2D cultured astrocytes. OPEN PRACTICES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Read the Editorial Highlight for this article on page 436.
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Acuaporina 4/metabolismo , Astrocitos/efectos de los fármacos , Proteínas de Unión al Calcio/metabolismo , Distroglicanos/metabolismo , Laminina/farmacología , Proteínas de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Animales , Astrocitos/metabolismo , Células Cultivadas , Quinasa 1 de Adhesión Focal/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/efectos de los fármacos , ARN Interferente Pequeño/farmacología , TransfecciónRESUMEN
Cisplatin (CDDP) is widely used as an anti-cancer platinum agent but its therapeutic efficacy is limited by acquired drug resistance. To develop a new therapeutic strategy that could overcome this resistance, it is important to characterize CDDP-resistant cancer cells. Here we established human lung cancer A549 cell-derived low- and high-grade CDDP-resistant sublines, termed ACR4 and ACR20â¯cells, by stepwise increasing CDDP concentrations up to 4 and 20⯵M, respectively. ACR4 and ACR20â¯cells showed 6- and 16-fold higher resistance to CDDP than A549â¯cells, respectively. Cell migration, invasion, and sphere formation were significantly decreased, whereas expression of the stem cell marker CD44v was increased in order of A549, ACR4, and ACR20â¯cells. The expression of the cystine-glutamate transporter xCT, which is encoded by SLC7A11, was upregulated because of the increased cell surface expression of CD44v in ACR20â¯cells. Treatment with the xCT inhibitor salazosulfapyridine and knockdown of SLC7A11 mRNA by a specific siRNA significantly improved sensitivity to CDDP in A549, ACR4, and ACR20â¯cells. Thus, our results suggest that CD44v overexpression is not involved in cancer stem cell properties but increases xCT expression, which leads to the acquisition of CDDP-resistance. This mechanism may contribute to the development of a new therapeutic strategy that can overcome resistance.
Asunto(s)
Sistema de Transporte de Aminoácidos y+/genética , Cisplatino/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Receptores de Hialuranos/metabolismo , Neoplasias Pulmonares/genética , Regulación hacia Arriba , Células A549 , Sistema de Transporte de Aminoácidos y+/metabolismo , Biomarcadores de Tumor/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Humanos , Concentración 50 Inhibidora , Neoplasias Pulmonares/patología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
OBJECTIVE: UVB irradiation is an established treatment for immunoinflammatory cutaneous disorders and has been shown to suppress cutaneous and systemic inflammatory diseases through modulation of the adaptive immune response. However, it remains unknown whether UVB irradiation prevents an immunoinflammatory disease of arteries such as atherosclerosis. APPROACH AND RESULTS: Here, we show that UVB exposure inhibits the development and progression of atherosclerosis in atherosclerosis-prone mice by expanding and enhancing the functional capacity of CD4+ forkhead box P3+ regulatory T cells and regulating proatherogenic T-cell responses. Experimental studies in Langerhans cell-depleted mice revealed that epidermal Langerhans cells play a critical role in UVB-dependent induction of CD4+ forkhead box P3+ regulatory T cells, suppression of proatherogenic T-cell responses, and prevention of atherosclerotic plaque development. CONCLUSIONS: Our findings suggest the skin immune system as a novel therapeutic target for atherosclerosis and provide a novel strategy for the treatment and prevention of atherosclerosis.
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Aorta/efectos de la radiación , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Inflamación/prevención & control , Piel/efectos de la radiación , Linfocitos T Reguladores/efectos de la radiación , Rayos Ultravioleta , Animales , Aorta/inmunología , Aorta/metabolismo , Aorta/patología , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Células Cultivadas , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Predisposición Genética a la Enfermedad , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Células de Langerhans/inmunología , Células de Langerhans/metabolismo , Células de Langerhans/efectos de la radiación , Activación de Linfocitos/efectos de la radiación , Ratones Noqueados , Fenotipo , Placa Aterosclerótica , Transducción de Señal/efectos de la radiación , Piel/inmunología , Piel/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismoRESUMEN
BACKGROUND: Gut microbiome composition or circulating microbiome-related metabolites in patients with heart failure (HF) have not been investigated at different time points (i.e., in the decompensated (Decomp) and compensated (Comp) phases). MethodsâandâResults: We prospectively enrolled 22 patients admitted for HF and 11 age-, sex-, and comorbidity-matched hospitalized control subjects without a history of HF. Gut flora and plasma microbiome-related metabolites were evaluated by amplicon sequencing of the bacterial 16S ribosomal RNA gene and capillary electrophoresis time-of-flight mass spectrometry, respectively. HF patients were evaluated in both the Decomp and Comp phases during hospitalization. The phylum Actinobacteria was enriched in HF patients compared with control subjects. At the genus level, Bifiodobacterium was abundant while Megamonas was depleted in HF patients. Meanwhile, plasma concentration of trimethylamine N-oxide (TMAO), a gut microbiome-derived metabolite, was increased in HF patients (Decomp HF vs. control, P=0.003; Comp HF vs. control, P=0.004). A correlation analysis revealed positive correlations between the abundance of the genus Escherichia/Shigella and levels of TMAO and indoxyl sulfate (IS, a microbe-dependent uremic toxin) in Comp HF (TMAO: r=0.62, P=0.002; IS: r=0.63, P=0.002). Escherichia/Shigella was more abundant in Decomp than in Comp HF (P=0.030). CONCLUSIONS: Our results suggest that gut microbiome composition and microbiome-related metabolites are altered in HF patients.
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Bifidobacterium , Escherichia coli , Microbioma Gastrointestinal , Insuficiencia Cardíaca , Shigella , Anciano , Anciano de 80 o más Años , Bifidobacterium/clasificación , Bifidobacterium/aislamiento & purificación , Escherichia coli/clasificación , Escherichia coli/aislamiento & purificación , Femenino , Insuficiencia Cardíaca/sangre , Insuficiencia Cardíaca/microbiología , Insuficiencia Cardíaca/terapia , Humanos , Masculino , Persona de Mediana Edad , Shigella/clasificación , Shigella/aislamiento & purificaciónRESUMEN
The gut microbiota were shown to play critical roles in the development of atherosclerosis, but the detailed mechanism is limited. The purpose of this study is to clarify the influence of gut microbiota on atherogenesis via lipid metabolism and systemic inflammation. Germ-free or conventionally raised (Conv) ApoE-deficient (ApoE-/-) mice were fed chow diet and euthanized at 20 weeks of age. We found that the lack of gut microbiota in ApoE-/- mice caused a significant increase in the plasma and hepatic cholesterol levels compared with Conv ApoE-/- mice. The absence of gut microbiota changed the bile acid composition in the ileum, which was associated with activation of the enterohepatic fibroblast growth factor 15, fibroblast growth factor receptor 4 axis, and reduction of cholesterol 7α-hydroxylase and hepatic bile acid synthesis, resulting in the accumulation of liver cholesterol content. However, we found that the lack of microbiota caused a significant reduction in atherosclerotic lesion formation compared with Conv ApoE-/- mice, which might be associated with the attenuation of lipopolysaccharide-mediated inflammatory responses. Our findings indicated that the gut microbiota affected both hypercholesterolemia and atherogenesis in mice.
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Apolipoproteínas E/genética , Aterosclerosis/microbiología , Colesterol/metabolismo , Inflamación/microbiología , Animales , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Bacterias/metabolismo , Bacterias/patogenicidad , Colesterol/genética , Colesterol 7-alfa-Hidroxilasa/genética , Dieta , Modelos Animales de Enfermedad , Factores de Crecimiento de Fibroblastos/genética , Microbioma Gastrointestinal/genética , Homeostasis , Humanos , Íleon/metabolismo , Íleon/microbiología , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Metabolismo de los Lípidos/genética , Ratones , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genéticaRESUMEN
We have reported that knockdown of Necl-4 decreases vascular endothelial growth factor (VEGF)-induced phosphorylation of extracellular signal-regulated kinase (ERK) without affecting phosphorylation of VEGF receptor 2 (VEGFR2) in sparsely cultured human umbilical vein endothelial cells (HUVECs). However, the underlying molecular mechanism is unknown. Compared with control HUVECs, VEGF-induced phosphorylation of phospholipase Cγ (PLCγ), c-Raf, mitogen-activated protein kinase/ERK kinase (MEK) and ERK were all inhibited in Necl-4-knockdown HUVECs. However, VEGF-induced internalization of VEGFR2 was not different between control and Necl-4-knockdown HUVECs. We have reported that protein-tyrosine phosphatase, non-receptor type 13 (PTPN13) and Rho-associated kinase (ROCK) are involved in the Necl-4-knockdown-induced inhibition of the VEGF-induced activation of Rac1. However, the effects of Necl-4-knockdown on VEGF-induced phosphorylation of VEGFR2 and ERK were not affected either by knockdown of PTPN13 or by ROCK inhibitors. These results suggest that Necl-4 enhances VEGF-induced activation of PLCγ-c-Raf-MEK-ERK pathway without affecting the phosphorylation and internalization of VEGFR2.
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Moléculas de Adhesión Celular/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Inmunoglobulinas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfolipasa C gamma/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Células Cultivadas , HumanosRESUMEN
N-glycosylation of proteins is important for protein folding and function. We have recently reported that FAM5C/BRINP3 contributes to the tumor necrosis factor-α-induced expression of leukocyte adhesion molecules in vascular endothelial cells (ECs). However, regulatory mechanism of the FAM5C biosynthesis is poorly understood. Co-immunoprecipitation assay revealed the interaction of FAM5C with UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1), a glycoprotein folding-sensor enzyme. FAM5C ectopically expressed in HEK293 cells was localized to the endoplasmic reticulum and co-localized with endogenously expressed UGGT1. Molecular size of FAM5C was reduced by treatment with N-glycosidase F and in FAM5C-expressing cells cultured in the presence of the N-glycosylation inhibitor tunicamycin. FAM5C was secreted by the cells and the secretion of FAM5C was blocked by tunicamycin. Among six potential N-glycosylation sites, the potential site at Asn168 was not N-glycosylated, and Asn337, Asn456, Asn562, Asn609, and Asn641 mutants were poorly secreted by the cells. These results demonstrated that FAM5C is an N-glycosylated protein and N-glycosylation is necessary for the secretion of FAM5C.
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Asparagina/metabolismo , Proteínas de Unión al ADN/metabolismo , Glucosiltransferasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Unión al ADN/genética , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Expresión Génica , Glucosiltransferasas/genética , Glicosilación/efectos de los fármacos , Células HEK293 , Humanos , Mutación , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Pliegue de Proteína , Tunicamicina/farmacologíaRESUMEN
BACKGROUND: Previously, we have reported that daily glucose fluctuations could affect coronary plaque vulnerability, but the underlying mechanisms remained unclear. This study sought to investigate the impact of CD14++CD16+ monocytes on plaque vulnerability, as assessed by virtual histology intravascular ultrasound (VH-IVUS), as well as their relationship to fluctuating glucose levels in patients with asymptomatic coronary artery disease (CAD). METHODS: Fifty-one patients with asymptomatic CAD, who were undergoing lipid-lowering therapy and underwent VH-IVUS evaluation for angiographically mild to moderate lesions, were enrolled in the study. Standard VH-IVUS parameters, including the percentage volume of the necrotic core (%NC) within the plaque and the presence of a virtual histology thin-cap fibroatheroma (VH-TCFA), were then evaluated. Additionally, monocyte subsets were assessed by flow cytometry, and daily glucose fluctuations were analyzed by measuring the mean amplitude of glycemic excursion (MAGE). RESULTS: Among 82 plaques from 22 diabetes mellitus (DM) patients and 29 non-DM patients, 15 VH-TCFAs were identified. CD14++CD16+ monocyte counts significantly correlated with both %NC and the presence of VH-TCFA (%NC: r = 0.339, p = 0.002; VH-TCFA: p = 0.003). Multivariate logistic regression analysis revealed that CD14++CD16+ monocyte counts were independently associated with VH-TCFA (odds ratio = 1.029, p = 0.004). Furthermore, CD14++CD16+ monocyte counts were significantly correlated with the MAGE score in the non-DM patients (r = 0.544, p = 0.005). CONCLUSIONS: CD14++CD16+ monocyte levels are associated with coronary plaque vulnerability and can serve as a biomarker for VH-TCFA in patients with CAD undergoing lipid-lowering therapy. In patients without DM, glucose fluctuations may alter the balance of monocyte subsets. Trial registration UMIN Registry number: UMIN000021228.
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Enfermedad de la Arteria Coronaria/patología , Vasos Coronarios/diagnóstico por imagen , Monocitos/patología , Placa Aterosclerótica/patología , Anciano , Anciano de 80 o más Años , Angiografía Coronaria/métodos , Estudios Transversales , Diabetes Mellitus/inmunología , Diabetes Mellitus/patología , Femenino , Humanos , Receptores de Lipopolisacáridos/inmunología , Masculino , Persona de Mediana Edad , Receptores de IgG/inmunología , Factores de Riesgo , Ultrasonografía IntervencionalRESUMEN
OBJECTIVE: Although T-cell-mediated chronic inflammation contributes to atherosclerosis development, the role of a negative regulatory molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) in atherosclerosis is poorly understood. We investigated the effects of CTLA-4 overexpression on atherosclerosis in apolipoprotein E-deficient (Apoe(-/-)) mice. APPROACH AND RESULTS: We generated CTLA-4 transgenic (CTLA-4-Tg)/Apoe(-/-) mice that display constitutive cell surface and intracellular expression of CTLA-4 in T cells and assessed atherosclerosis at age 16 weeks. CTLA-4 overexpression significantly reduced atherosclerotic lesion formation and intraplaque accumulation of macrophage and CD4(+) T cells in the aortic root compared with controls. CTLA-4-Tg/Apoe(-/-) mice showed decreased numbers of effector CD4(+) T cells and decreased expression of costimulatory molecules CD80 and CD86, ligands for CTLA-4, and a costimulatory molecule CD28, on CD11c(+) dendritic cells compared with controls. Consistent with in vivo findings, in vitro experiments revealed that CD4(+) T cells from CTLA-4-Tg/Apoe(-/-) mice showed decreased proliferative capacity and proinflammatory cytokine production, downregulated CD80 expression on CD11c(+) dendritic cells, and suppressed the proliferation of other T cells by limiting the costimulatory pathway. Moreover, CD11c(+) dendritic cells from CTLA-4-Tg/Apoe(-/-) mice showed reduced proliferative activity of T cells in vitro, suggesting the suppression of dendritic cell maturation in vivo. CONCLUSIONS: CTLA-4 regulates atherosclerosis by suppressing proatherogenic immune responses and could be an attractive therapeutic target for atherosclerosis.
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Aorta/metabolismo , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Antígeno CTLA-4/metabolismo , Linfocitos T/metabolismo , Animales , Aorta/inmunología , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/metabolismo , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Antígeno CD11c/metabolismo , Antígenos CD28/metabolismo , Antígeno CTLA-4/genética , Proliferación Celular , Células Cultivadas , Células Dendríticas/metabolismo , Femenino , Genotipo , Activación de Linfocitos , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fenotipo , Placa Aterosclerótica , Transducción de Señal , Linfocitos T/inmunología , Regulación hacia ArribaRESUMEN
AIMS: A recent large clinical study demonstrated the association between intermediate CD14++CD16+monocytes and cardiovascular events. However, whether that monocyte subset contributes to the pathogenesis of atrial fibrillation (AF) has not been clarified. We compared the circulating monocyte subsets in AF patients and healthy people, and investigated the possible role of intermediate CD14++CD16+monocytes in the pathophysiology of AF. METHODS AND RESULTS: This case-control study included 44 consecutive AF patients without systemic diseases referred for catheter ablation at our hospital, and 40 healthy controls. Patients with systemic diseases, including structural heart disease, hepatic or renal dysfunction, collagen disease, malignancy, and inflammation were excluded. Monocyte subset analyses were performed (three distinct human monocyte subsets: classical CD14++CD16-, intermediate CD14++CD16+, and non-classical CD14+CD16++monocytes). We compared the monocyte subsets and evaluated the correlation with other clinical findings. A total of 60 participants (30 AF patients and 30 controls as an age-matched group) were included after excluding 14 AF patients due to inflammation. Atrial fibrillation patients had a higher proportion of circulating intermediate CD14++CD16+monocytes than the controls (17.0 ± 9.6 vs. 7.5 ± 4.1%, P < 0.001). A multivariable logistic regression analysis demonstrated that only the proportion of intermediate CD14++CD16+monocytes (odds ratio: 1.316; 95% confidence interval: 1.095-1.582, P = 0.003) was independently associated with the presence of AF. Intermediate CD14++CD16+monocytes were negatively correlated with the left atrial appendage flow during sinus rhythm (r= -0.679, P = 0.003) and positively with the brain natriuretic peptide (r = 0.439, P = 0.015). CONCLUSION: Intermediate CD14++CD16+monocytes might be closely related to the pathogenesis of AF and reflect functional remodelling of the left atrium.
Asunto(s)
Fibrilación Atrial/sangre , Función del Atrio Izquierdo , Remodelación Atrial , Receptores de Lipopolisacáridos/sangre , Monocitos/inmunología , Receptores de IgG/sangre , Anciano , Fibrilación Atrial/diagnóstico , Fibrilación Atrial/inmunología , Fibrilación Atrial/fisiopatología , Biomarcadores/sangre , Estudios de Casos y Controles , Distribución de Chi-Cuadrado , Femenino , Proteínas Ligadas a GPI/sangre , Humanos , Japón , Modelos Logísticos , Masculino , Persona de Mediana Edad , Monocitos/clasificación , Análisis Multivariante , Péptido Natriurético Encefálico/sangre , Oportunidad Relativa , Valor Predictivo de las Pruebas , Regulación hacia ArribaRESUMEN
The association between atherosclerosis and gut microbiota has been attracting increased attention. We previously demonstrated a possible link between gut microbiota and coronary artery disease. Our aim of this study was to clarify the gut microbiota profiles in coronary artery disease patients using data mining analysis of terminal restriction fragment length polymorphism (T-RFLP). This study included 39 coronary artery disease (CAD) patients and 30 age- and sex- matched no-CAD controls (Ctrls) with coronary risk factors. Bacterial DNA was extracted from their fecal samples and analyzed by T-RFLP and data mining analysis using the classification and regression algorithm. Five additional CAD patients were newly recruited to confirm the reliability of this analysis. Data mining analysis could divide the composition of gut microbiota into 2 characteristic nodes. The CAD group was classified into 4 CAD pattern nodes (35/39 = 90 %), while the Ctrl group was classified into 3 Ctrl pattern nodes (28/30 = 93 %). Five additional CAD samples were applied to the same dividing model, which could validate the accuracy to predict the risk of CAD by data mining analysis. We could demonstrate that operational taxonomic unit 853 (OTU853), OTU657, and OTU990 were determined important both by the data mining method and by the usual statistical comparison. We classified the gut microbiota profiles in coronary artery disease patients using data mining analysis of T-RFLP data and demonstrated the possibility that gut microbiota is a diagnostic marker of suffering from CAD.
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Enfermedad de la Arteria Coronaria/diagnóstico , Enfermedad de la Arteria Coronaria/microbiología , ADN Bacteriano/genética , Microbioma Gastrointestinal , Polimorfismo de Longitud del Fragmento de Restricción , Anciano , Biomarcadores , Estudios de Casos y Controles , Minería de Datos , Heces/microbiología , Femenino , Humanos , Japón , Masculino , Persona de Mediana EdadRESUMEN
The intestinal microbiota appears to play an important role in the development of atherosclerosis. We investigated the effect of the probiotic lactic acid bacterium Pediococcus acidilactici R037 on atherosclerosis using apolipoprotein E-deficient (ApoE -/-) mice. Six-week-old ApoE -/- mice were orally administered R037 six times a week. Mice treated with R037 for 12 weeks exhibited markedly attenuated atherosclerotic lesions in the aortic root (2.3 ± 0.15 × 105 µm2 vs. 3.3 ± 0.29 × 105 µm2, respectively; P < 0.01; n = 15-17 each group). The expression of Ki-67 in CD4+ T cells, the population of interferon γ-producing CD4+ T cells in the spleen, and pro-inflammatory cytokine production from splenic lymphocytes were significantly decreased in R037-treated mice. Interestingly, splenic dendritic cells (DCs) isolated from R037-treated mice suppressed CD4+ T-cell proliferation and pro-inflammatory cytokine production ex vivo, suggesting that R037 treatment induced tolerogenic DCs. Programmed cell death ligand 1 expression in DCs was significantly enhanced in R037-treated mice, which might explain the immunosuppressive effect of DCs at least in part. These results indicate that R037 attenuates atherosclerosis by inducing tolerogenic DCs, which suppress Th1-driven inflammation and the proliferative activity of CD4+ T cells. Our findings may provide a novel therapeutic approach for the prevention of atherosclerosis based on dietary supplementation with probiotics.
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Aterosclerosis/prevención & control , Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/inmunología , Pediococcus acidilactici , Probióticos/administración & dosificación , Administración Oral , Animales , Aorta/patología , Femenino , Ratones , Ratones Noqueados para ApoERESUMEN
Atherosclerosis is a chronic inflammatory disorder involving innate and adaptive immunity process. Effector T cell (Teff) responses promote atherosclerotic disease, whereas regulatory T cells (Tregs) have been shown to play a protective role against atherosclerosis by down-regulating inflammatory responses which include multiple mechanisms. Compelling experimental data suggest that shifting the Treg/Teff balance toward Tregs may be a possible therapeutic approach for atherosclerotic disease, although the role of Tregs in human atherosclerotic disease has not been fully elucidated. In this review, we discuss recent advances in our understanding of the roles of Tregs and Teffs in experimental atherosclerosis, as well as human coronary artery disease.