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OBJECTIVES: In order to assist junior doctors in better diagnosing apical periodontitis (AP), an artificial intelligence AP grading system was developed based on deep learning (DL) and its reliability and accuracy were evaluated. METHODS: 120 cone-beam computed tomography (CBCT) images were selected to construct a classification dataset with four categories, which were divided by CBCT periapical index (CBCTPAI), including normal periapical tissue, CBCTPAI 1-2, CBCTPAI 3-5 and young permanent teeth. Three classic algorithms (ResNet50/101/152) as well as one self-invented algorithm (PAINet) were compared with each other. PAINet were also compared with two recent Transformer-based models and three attention models. Their performance was evaluated by accuracy, precision, recall, balanced F score (F1-score) and the area under the macro-average receiver operating curve (AUC). Reliability was evaluated by Cohen's kappa ââto compare the consistency of model predicted labels with expert opinions. RESULTS: PAINet performed best among the four algorithms. The accuracy, precision, recall, F1-score and AUC on the test set were 0.9333, 0.9415, 0.9333, 0.9336 and 0.9972, respectively. Cohen's kappa was 0.911, which represented almost perfect consistency. CONCLUSIONS: PAINet can accurately distinguish between normal periapical tissues, CBCTPAI 1-2, CBCTPAI 3-5 and young permanent teeth. Its results were highly consistent with expert opinions. It can help junior doctors diagnose and score AP, reducing the burden. It can also be promoted in areas where experts are lacking to provide professional diagnostic opinions.
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Chromatin remodeler ARID1A regulates gene transcription by modulating nucleosome positioning and chromatin accessibility. While ARID1A-mediated stage and lineage-restricted gene regulation during cell fate canalization remains unresolved. Using osteoclastogenesis as a model, we show that ARID1A transcriptionally safeguards the osteoclast (OC) fate canalization during proliferation-differentiation switching at single-cell resolution. Notably, ARID1A is indispensable for the transcriptional apparatus condensates formation with coactivator BRD4/lineage-specifying transcription factor (TF) PU.1 at Nfatc1 super-enhancer during safeguarding the OC fate canalization. Besides, the antagonist function between ARID1A-cBAF and BRD9-ncBAF complex during osteoclastogenesis has been validated with in vitro assay and compound mutant mouse model. Furthermore, the antagonistic function of ARID1A-"accelerator" and BRD9-"brake" both depend on coactivator BRD4-"clutch" during osteoclastogenesis. Overall, these results uncover sophisticated cooperation between chromatin remodeler ARID1A, coactivator, and lineage-specifying TF at super-enhancer of lineage master TF in a condensate manner, and antagonist between distinct BAF complexes in the proper and balanced cell fate canalization.
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Diferenciación Celular , Linaje de la Célula , Proteínas de Unión al ADN , Osteoclastos , Osteogénesis , Factores de Transcripción , Animales , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Osteoclastos/metabolismo , Osteoclastos/citología , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Ratones , Osteogénesis/genética , Osteogénesis/fisiología , Factores de Transcripción NFATC/metabolismo , Factores de Transcripción NFATC/genética , Ensamble y Desensamble de Cromatina , Regulación de la Expresión Génica , Ratones Endogámicos C57BL , Proliferación Celular , Análisis de la Célula Individual , Proteínas que Contienen Bromodominio , Proteínas NuclearesRESUMEN
Reperfusion therapy after myocardial infarction may lead to myocardial injury, which can be complicated and exacerbated by diabetes. The existing therapeutic methods for myocardial ischemia-reperfusion injury (MIRI) in diabetic patients are not ideal. Oleoylethanolamide (OEA) has been found to have protective effects on diabetes and acute cerebral ischemia. This study aimed to determine whether OEA can alleviate MIRI in diabetic rats, and to explore the underlying mechanism. The model of diabetic rats with MIRI was established by blocking the left coronary artery for 30 min, followed by restoring blood flow stability for 120 min. The myocardial enzyme spectrum, area of MIRI, and expression levels of apoptosis-related proteins were detected. The results showed that OEA pretreatment could reduce myocardial infarction area, protect myocardial tissue structure, and reduce myocardial cell apoptosis in diabetic rats with MIRI. Meanwhile, the levels of creatine kinase (CK)-MB (CK-MB), lactate dehydrogenase (LDH), and malondialdehyde (MDA) were reduced, while superoxide dismutase (SOD) level was elevated. H9C2 cells were treated with high glucose and oxygen-glucose deprivation/reperfusion (OGD/R) to establish an in vitro model. Capsazepine (CPZ), an antagonist of transient receptor potential vanilloid subtype 1 (TRPV1), and LY294002, an inhibitor of PI3K, were used to treat H9C2 cells in vitro. Apoptosis level and the expression levels of apoptosis-related proteins were measured. It was found that OEA activated TRPV1 and the PI3K/Akt signaling pathway, downregulated the expression levels of apoptosis-related proteins (Bcl-2 and cleaved caspase-3), and ameliorated the apoptosis of H9C2 cells treated with high glucose and OGD/R. This study clarified that OEA, as a TRPV1 agonist, could reduce myocardial cell apoptosis by activating the PI3K/Akt signaling pathway in diabetic rats with MIRI. The findings may provide a theoretical basis for administration of OEA as a potential therapeutic agent into diabetic patients with MIRI.
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BACKGROUND: Obesity is the cause of multiple metabolic disorders, and its incidence has been rapidly increasing worldwide. It develops when energy intake exceeds energy expenditure (EE). Wedelolactone (WDL) is a naturally isolated compound from Eclipta prostrata L. and possesses many pharmacological activities. However, little is known about the effect of WDL on obesity and EE. PURPOSE: The present study aimed to investigate the effect of WDL on obesity and EE in diet-induced obese (DIO) mice and its underlying mechanism. METHODS: Obese mice were induced by high fat diet. The effects of WDL on obese mice were assessed by examining body weight, fat mass, EE, glucose tolerance, and hepatic and kidney injury. 3T3-L1 cells were differentiated into mature adipocytes and incubated with WDL in vitro. Immunohistochemistry, western blotting, and real-time PCR were used to assess adipose browning. The inhibitory efficiency of WDL on nicotinamide N-methyltransferase (NNMT) was evaluated using a fluorescence assay. RESULTS: WDL reduced fat mass, suppressed body weight gain, and improved obesity-related metabolic disorders in DIO mice. WDL treatment promoted adipose browning and enhanced EE in both DIO mice and 3T3-L1 cells. These effects were eliminated in AMPK antagonized or PPARα knockdown cells and in PPARα-/- mice. Furthermore, we identified the target of WDL to be NNMT, an appealing target for regulating energy metabolism. WDL inhibited NNMT with an extremely low IC50 of 0.03 µM. Inhibition of NNMT and activation of SIRT1/AMPK/PPARα explains how WDL reverses obesity by prompting adipose browning. CONCLUSION: Our findings demonstrate the novel effects of WDL in promoting adipose browning, enhancing EE and attenuating obesity and uncover the underlying mechanism, which includes inhibition of NNMT and subsequently activation of SIRT1/AMPK/PPARα in response to WDL. WDL could be further developed as a therapeutic agent for treating obesity and related metabolic diseases.
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Nicotinamida N-Metiltransferasa , Sirtuina 1 , Células 3T3-L1 , Proteínas Quinasas Activadas por AMP , Animales , Cumarinas , Dieta Alta en Grasa , Ratones , Ratones Endogámicos C57BL , Obesidad/tratamiento farmacológico , PPAR alfa , FitoquímicosRESUMEN
Oleoylethanolamide (OEA) is an endogenous peroxisome proliferator-activated receptor α (PPARα) agonist that acts on the peripheral control of energy metabolism. However, its therapeutic potential and related mechanisms in hepatic glucose metabolism under type 2 diabetes mellitus (T2DM) are not clear. Here, OEA treatment markedly improved glucose homeostasis in a PPARα-independent manner. OEA efficiently promoted glycogen synthesis and suppressed gluconeogenesis in mouse primary hepatocytes and liver tissue. OEA enhanced hepatic glycogen synthesis and inhibited gluconeogenesis via liver kinase B1 (LKB1)/5' AMP-activated protein kinase (AMPK) signaling pathways. PPARα was not involved in the roles of OEA in the LKB1/AMPK pathways. We found that OEA exerts its antidiabetic effect by increasing glycogenesis and decreasing gluconeogenesis via the LKB1/AMPK pathway. The ability of OEA to control hepatic LKB1/AMPK pathways may serve as a novel therapeutic approach for the treatment of T2DM. SIGNIFICANCE STATEMENT: Oleoylethanolamide (OEA) exerted a potent antihyperglycemic effect in a peroxisome proliferator-activated receptor α-independent manner. OEA played an antihyperglycemic role primarily via regulation of hepatic glycogen synthesis and gluconeogenesis. The main molecular mechanism of OEA in regulating liver glycometabolism is activating the liver kinase B1/5' AMP-activated protein kinase signaling pathways.
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Diabetes Mellitus Tipo 2/sangre , Endocannabinoides/farmacología , Gluconeogénesis/fisiología , Glucógeno/biosíntesis , Hígado/metabolismo , Ácidos Oléicos/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP , Animales , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 2/inducido químicamente , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Endocannabinoides/uso terapéutico , Gluconeogénesis/efectos de los fármacos , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Ácidos Oléicos/uso terapéutico , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Oleoylethanolamide (OEA) is an endogenous lipid mediator involved in the control of feeding, body weight, and energy metabolism. However, whether OEA modulates maturation of dendritic cells (DCs) has never been addressed. Hence, we evaluated the effect of OEA on DCs maturation in bone marrow-derived DCs (BMDCs) in four aspects: (a) Cell surface markers were determined using flow cytometric analysis; (b) cell mobile ability was testified with the transwell assay; (c) stimulation of T cells proliferation was performed in a coculture system; and (d) cytokine production was measured using polymerase chain reaction (PCR). The result showed that, in mature BMDCs induced by lipopolysaccharides (LPS), the OEA treatment decreased expressions of cell surface markers, reduced cell migration, diminished the proliferation of cocultured T cells, and regulated cytokine production of BMDCs, indicating the modulatory effect of OEA on DCs maturation. Furthermore, to explore the underlying mechanism of the immunomodulatory effect of OEA, we used antagonists of transient receptor potential vanilloid-1 (TRPV1) and AMP-activated protein kinase (AMPK), determined the protein expressions of TRPV1/AMPK and Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) using western blot, and measured the intracellular calcium concentration using calcium imaging. The result illustrated that OEA downregulated TLR4/NF-κB, the classical pathway leading to DCs maturation induced by LPS, through the activation of TRPV1 and AMPK. Collectively, the present study suggests that OEA suppresses DCs maturation through the activation of TRPV1/AMPK. These findings increase our understanding of this endogenous lipid OEA.
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Proteínas Quinasas Activadas por AMP/metabolismo , Células Dendríticas/efectos de los fármacos , Endocannabinoides/farmacología , Factores Inmunológicos/farmacología , Ácidos Oléicos/farmacología , Canales Catiónicos TRPV/metabolismo , Animales , Calcio/metabolismo , Células Dendríticas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Femenino , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/metabolismoRESUMEN
OBJECTIVE: Tongxinluo (TXL) is a traditional Chinese medicine (TCM). It is used to treat coronary heart disease and atherosclerosis. We investigated the effects of TXL on the neointima formation and expression of inflammatory cytokines in rats after carotid artery balloon injury. MATERIALS AND METHODS: Male Sprague-Dawley rats were randomly divided into four groups: sham operation group (Sham, n = 15), balloon injury group treated with vehicle (Control, n = 15), TXL low-dose group treated with TXL of 0.5 g/kg/d (TXL-L, n = 15), and TXL high-dose group treated with TXL of 1.0 g/kg/d (TXL-H, n = 15). TXL was given by gavage daily. 14 days after injury', the levels of serum nitric oxide (NO), endothelin-1 (ET-1), monocyte chemoattractant protein-1 (MCP-1), and soluble intercellular adhesion molecule-1 (sICAM-1) were evaluated. The morphology of carotid artery tissue was observed with hematoxylin-eosin staining. Expressions of MCP-1 and ICAM-1 in the artery were detected by real-time polymerase chain reaction (RT-PCR) and western blotting. RESULTS: 14 days after injury, a significant increase in concentrations of serum ET-1, MCP-1, and sICAM-1 (P < 0.05), as well as a significant decrease in NO serum level were observed in rats subjected to artery injury compared to the sham rats (P < 0.05). TXL significantly decreased ET-1, MCP-1 and sICAM-1 serum levels (P < 0.05), whereas significantly increased NO serum level compared with the control (P < 0.05). TXL significantly reduced the neointimal thickening at day14 after injury (P < 0.05). In addition, TXL significantly reduced mRNA and protein expressions of ICAM-1 and MCP-1 in injured artery (P < 0.05). CONCLUSIONS: This study demonstrates that TXL is effective in improving endothelial function, attenuating neointimal formation of artery after balloon injury, and reducing expression of inflammatory cytokine MCP-1 and ICAM-1. It may be a useful agent for protecting the artery against injury.
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Traumatismos de las Arterias Carótidas/tratamiento farmacológico , Medicamentos Herbarios Chinos/farmacología , Neointima/prevención & control , Animales , Western Blotting , Traumatismos de las Arterias Carótidas/patología , Quimiocina CCL2/sangre , Quimiocina CCL2/efectos de los fármacos , Quimiocina CCL2/genética , Citocinas/efectos de los fármacos , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Medicamentos Herbarios Chinos/administración & dosificación , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Regulación de la Expresión Génica/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Molécula 1 de Adhesión Intercelular/sangre , Molécula 1 de Adhesión Intercelular/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/genética , Masculino , ARN Mensajero , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
AIM: To investigate the mechanisms underlying the pro-angiogenic effects of statin, the effects of atorvastatin were investigated on the expression of angiogenic factors in ischemic hindlimbs of rats. The function and number of endothelial progenitor cells (EPCs) were investigated in hypertensive rats. METHODS: Hindlimb ischemia rats were administered 10 or 30 mg/kg/day atorvastatin orally for 2 weeks. Angiogenesis was evaluated by a laser Doppler and by Isolectin-B4 immunostaining. The expressions of VEGF, IL-8, angiopoietin (Ang)-1, Ang-2, eNOS, and hemoxidase (HO)-1 were evaluated by Western blotting and immunohistochemistry. Spontaneously hypertensive rats (SHR) were administered 10 mg/kg/day atorvastatin. EPC function was evaluated by colony formation and migration. The EPC number was evaluated by CD34-positive cells. RESULTS: A lowdose of atorvastatin, but not a highdose, significantly increased regional blood flow. Atorvastatin significantly increased the expressions of VEGF, IL-8, Ang-1, Ang-2, eNOS, and HO-1 proteins in ischemic hindlimbs. Atorvastatin significantly increased the number and colony formation of EPCs and decreased oxidation in mononuclear cells from SHR. CONCLUSION: Atorvastatin strongly induced angiogenesis with increases in angiogenic cytokines, HO-1 and EPC numbers. Statins are thus considered potertial agents for therapeutic angiogenesis.
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Células Endoteliales/fisiología , Ácidos Heptanoicos/farmacología , Miembro Posterior/irrigación sanguínea , Isquemia/tratamiento farmacológico , Neovascularización Fisiológica/efectos de los fármacos , Pirroles/farmacología , Células Madre/fisiología , Inductores de la Angiogénesis/administración & dosificación , Inductores de la Angiogénesis/farmacología , Proteínas Angiogénicas/análisis , Animales , Atorvastatina , Recuento de Células , Endotelio Vascular/citología , Ácidos Heptanoicos/administración & dosificación , Isquemia/patología , Pirroles/administración & dosificación , Ratas , Ratas Endogámicas SHR , Células Madre/citologíaRESUMEN
Pyrrole-imidazole polyamide can be combined in antiparallel side-by-side dimeric complexes along the minor groove of DNA in a sequence-specific manner. Pyrrole-imidazole polyamides are effective inhibitors of transcription factors as well as viral repressors and transactivators. Recently, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) was reported to be a major factor contributing to the pathogenesis of coronary atherosclerosis. In this study, we designed a pyrrole-imidazole polyamide specific for the LOX-1 gene and evaluated its effect on LOX-1 gene transcription. A pyrrole-imidazole polyamide was designed to target the AP-1 binding site of the LOX-1 gene and synthesized by solid phase methods. This pyrrole-imidazole polyamide significantly inhibited LOX-1 promoter activity in HEK293 cells, determined by the luciferase assay. LOX-1 mRNA expression was also inhibited by the pyrrole-imidazole polyamide at a concentration of 10-9 mol/l in human umbilical vein endothelial cells (HUVEC), determined by the real-time PCR method. HUVEC were treated by pyrrole-imidazole polyamide targeting the LOX-1 gene, and apoptosis was assessed using Hoechst stain, terminal deoxy nucleotidyl transferase-mediated UTP end labeling method, and dye-uptake bioassay. Treatment of HUVEC for 72 h with LOX-1 targeted pyrrole-imidazole polyamide decreased apoptosis induced by angiotensin II and oxidized low-density lipoprotein (ox-LDL) loading in all assays. This novel therapeutic agent, pyrrole-imidazole polyamide, could specifically inhibit LOX-1 gene expression by reducing the promoter activity of the gene. Pyrrole-imidazole polyamide seems to be a powerful promising new agent that can be used to explore therapies based on inhibition of transcription. Molecular recognition of DNA by small molecules could provide insight into the development of new human medicines.
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Imidazoles/farmacología , Lipoproteínas LDL/farmacología , Nylons/farmacología , Pirroles/farmacología , Receptores Depuradores de Clase E/genética , Apoptosis/efectos de los fármacos , Bencimidazoles/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Fluoresceína-5-Isotiocianato/metabolismo , Colorantes Fluorescentes/metabolismo , Silenciador del Gen/efectos de los fármacos , Marcación de Gen , Humanos , Imidazoles/síntesis química , Imidazoles/química , Etiquetado Corte-Fin in Situ , Riñón/citología , Lipoproteínas LDL/antagonistas & inhibidores , Luciferasas/metabolismo , Estructura Molecular , Nylons/síntesis química , Nylons/química , Oxidación-Reducción , Regiones Promotoras Genéticas/efectos de los fármacos , Pirroles/síntesis química , Pirroles/química , ARN Mensajero/metabolismo , Receptores Depuradores de Clase E/metabolismo , Factores de Tiempo , Venas Umbilicales/citologíaRESUMEN
AIMS: Although the use of drug-eluting stents (DESs) has been shown to limit neointima hyperplasia, currently available DESs may adversely affect re-endothelialization. To evaluate whether a novel gene silencer pyrrole-imidazole (PI) polyamide targeting transforming growth factor (TGF)-beta1 is a candidate agent for the DESs, we examined the effects of PI polyamide targeting the TGF-beta1 promoter on neointimal formation in rat carotid artery after balloon injury. METHODS AND RESULTS: PI polyamide was designed to span the boundary of the AP-1 binding site of the TGF-beta1 promoter. After inducing balloon injury to arteries, incubation with PI polyamide was carried out for 10 min. Neointimal thickening and re-endothelialization were evaluated at 21 days after injury. Fluoresceinisothiocyanate-labelled PI polyamide was distributed into most of the nuclei in the injured artery without any delivery reagents. PI polyamide (100 microg) significantly inhibited neointimal thickening at 21 days after injury by 57%. PI polyamide targeting TGF-beta1 significantly decreased the expression of TGF-beta1 mRNA and protein in the artery at 3 days after injury and also suppressed the expression of connective tissue growth factor (CTGF), fibronectin, collagen type 1, and lectin-like ox-LDL receptor-1 mRNAs. A morphometric analysis showed that PI polyamide targeting TGF-beta1 accelerated re-endothelialization in the injured artery. CONCLUSION: These findings suggest that the synthetic PI polyamide targeting the TGF-beta1 promoter may have the potential to suppress neointimal hyperplasia after arterial injury by the down-regulation of TGF-beta1 and CTGF and the reduction of the extracellular matrix. As a result, PI polyamide targeting TGF-beta1 may therefore be a potentially effective agent for the treatment of in-stent restenosis, as a candidate agent for the next-generation DES.
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Traumatismos de las Arterias Carótidas/tratamiento farmacológico , Estenosis Carotídea/tratamiento farmacológico , Endotelio Vascular/efectos de los fármacos , Imidazoles/farmacología , Nylons/farmacología , Regiones Promotoras Genéticas/efectos de los fármacos , Pirroles/farmacología , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/patología , Estenosis Carotídea/metabolismo , Estenosis Carotídea/patología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colágeno Tipo I/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Modelos Animales de Enfermedad , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Fibronectinas/metabolismo , Silenciador del Gen , Hiperplasia , Imidazoles/metabolismo , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Nylons/metabolismo , Pirroles/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas WKY , Receptores Depuradores de Clase E/metabolismo , Prevención Secundaria , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
The purpose of this study was to evaluate whether the spontaneously hypertensive rat SHRSP.Z-Lepr(fa)/IzmDmcr (SHRSP fatty) is a useful animal model to clarify molecular mechanisms that underlie metabolic syndrome. We investigated histopathologic changes in the cardiovascular organs and metabolic characteristics of SHRSP fatty rats, which are congenic rats from a cross between SHRSP and Zucker fatty (ZF) rats. The aortic wall and cardiac, carotid, and renal arteries from SHRSP and SHRSP fatty rats were thicker than those of ZF rats. The renal cortex in SHRSP and SHRSP fatty rats showed severe glomerulosclerosis. Pancreatic islands in SHRSP fatty and ZF rats showed marked hyperplasia. Steady-state plasma glucose concentrations were higher in SHRSP fatty than in ZF rats. Non-fasting triglyceride levels in SHRSP fatty rats were higher than in ZF rats. DNA synthesis in cultured vascular smooth muscle cells (VSMCs) from SHRSP fatty and SHRSP rats was significantly higher than that in VSMCs from Wistar-Kyoto (WKY) or ZF rats. Levels of platelet-derived growth factor A-chain and transforming growth factor-beta1 mRNAs were higher in VSMCs from SHRSP fatty and SHRSP than from ZF rats. Microarray analysis identified five genes that were significantly upregulated and four genes that were significantly downregulated in visceral adipose tissue of SHRSP fatty rats compared with levels in control strains (SHRSP and ZF rats). These findings suggest that the combination of hypertension and obesity accelerates vascular remodeling, dyslipidemia, and insulin resistance in metabolic syndrome. The phenotype of SHRSP fatty is similar to that of human metabolic syndrome, and therefore, studies of these rats may help clarify the molecular mechanisms that underlie metabolic syndrome in humans.
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Sistema Cardiovascular/metabolismo , Perfilación de la Expresión Génica , Hipertensión/metabolismo , Síndrome Metabólico/metabolismo , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Glucemia/metabolismo , Sistema Cardiovascular/patología , Células Cultivadas , Modelos Animales de Enfermedad , Epidídimo/metabolismo , Epidídimo/patología , Hipertensión/patología , Insulina/sangre , Resistencia a la Insulina , Corteza Renal/metabolismo , Corteza Renal/patología , Masculino , Síndrome Metabólico/patología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Páncreas/metabolismo , Páncreas/patología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas , Ratas Endogámicas WKY , Ratas Zucker , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
BACKGROUND: Endothelial progenitor cells (EPCs) derived from bone marrow migrate to areas of endothelial damage and repair them. EPC function is impaired by oxidative stress. We examined the effects of an antioxidative beta1-adrenoceptor blocker on the number and function of EPCs in hypertensive rats. METHODS: Spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats were fed diets loaded with high salt. The SHRs were treated with celiprolol or atenolol for 2 weeks. Peripheral blood mononuclear cells (MNCs) were separated, subjected to flow cytometric analysis to determine the number of circulating EPCs, and cultured to quantify EPC colony formation. EPC migration was evaluated in migration assay chambers. EPC senescence was evaluated using beta-galactosidase assay. Oxidative stress of EPCs was evaluated using thiobarbituric acid-reactive substance (TBARS) assay. The expression of nicotinamine adenine dinucleotide phosphate (NAD(P)H) oxidase component mRNAs in the renal cortex, aorta, and heart were evaluated by real-time PCR. RESULTS: The number, colony formation, and migration of EPCs in SHRs were significantly lower than those in WKY rats. TBARS scores in EPCs from SHRs were significantly higher than those from WKY rats. Celiprolol increased the number of circulating EPCs and stimulated EPC colony formation and migration, while decreasing EPC senescence. Celiprolol inhibited oxidation in EPCs from SHRs, and decreased the expression of NAD(P)H oxidase component mRNAs in the renal cortex, aorta, and heart. CONCLUSION: EPCs are impaired in SHRs in response to oxidative stress. Celiprolol decreases oxidative stress in hypertension in vivo and improves EPC numbers and function. It appears, therefore, that celiprolol may exert beneficial cardiovascular effects through its antioxidative properties.
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Antagonistas Adrenérgicos beta/farmacología , Antioxidantes/farmacología , Celiprolol/farmacología , Células Endoteliales/efectos de los fármacos , Hipertensión/tratamiento farmacológico , Células Madre/efectos de los fármacos , Animales , Movimiento Celular , Células Cultivadas , Masculino , NADPH Oxidasas/análisis , Estrés Oxidativo/efectos de los fármacos , ARN Mensajero/análisis , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKYRESUMEN
Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a membrane protein that can support the binding, internalization, and proteolytic degradation of oxidized low-density lipoprotein. The LOX-1 expression increases in the neointima after balloon injury. To develop an efficient compound to inhibit LOX-1, we designed and synthesized a novel gene silencer pyrrole-imidazole (PI) polyamide targeting the rat LOX-1 gene promoter (PI polyamide to LOX-1) to the activator protein-1 binding site. We examined the effects of PI polyamide to LOX-1 on the LOX-1 promoter activity, the expression of LOX-1 mRNA and protein, and neointimal hyperplasia of the rat carotid artery after balloon injury. PI polyamide to LOX-1 significantly inhibited the rat LOX-1 promoter activity and decreased the expression of LOX-1 mRNA and protein. After balloon injury of the arteries, PI polyamide to LOX-1 was incubated for 10 minutes. Fluorescein isothiocyanate-labeled PI polyamide was distributed to almost all of the nuclei in the injured artery. PI polyamide to LOX-1 (100 microg) significantly inhibited the neointimal thickening by 58%. PI polyamide preserved the re-endothelialization in the injured artery. PI polyamide significantly inhibited the expression of LOX-1, monocyte chemoattractant protein-1, intercellular adhesion molecule-1, and matrix metalloproteinase-9 mRNAs in the injured artery. The synthetic PI polyamide to LOX-1 decreased the expression of LOX-1 and inhibited neointimal hyperplasia after arterial injury. This novel gene silencer PI polyamide to LOX-1 is, therefore, considered to be a feasible agent for the treatment of in-stent restenosis.
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Reestenosis Coronaria/prevención & control , Expresión Génica/efectos de los fármacos , Nylons/farmacología , Pirroles/farmacología , Receptores Depuradores de Clase E/antagonistas & inhibidores , Túnica Íntima/efectos de los fármacos , Angioplastia de Balón/efectos adversos , Animales , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/patología , Arterias Carótidas/cirugía , Células Cultivadas , Reestenosis Coronaria/genética , Reestenosis Coronaria/patología , Diseño de Fármacos , Silenciador del Gen/efectos de los fármacos , Hiperplasia/genética , Hiperplasia/prevención & control , Masculino , Nylons/síntesis química , Nylons/química , Regiones Promotoras Genéticas/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Pirroles/síntesis química , Pirroles/uso terapéutico , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores Depuradores de Clase E/genética , Túnica Íntima/patología , Túnica Íntima/cirugíaRESUMEN
We have shown that spontaneously hypertensive rat (SHR)-derived vascular smooth muscle cells (VSMCs) change to the synthetic phenotype and show increased expression of complement 3 (C3) and that C3 plays a role in the change to the synthetic phenotype. To determine the mechanisms underlying the effects of C3 on this phenotypic change, we examined the effects of C3a on transcription factors involved in VSMC phenotype and found that C3a increased the expression of Krüppel-like zinc-finger transcription factor 5 (KLF5) mRNA. C3a increased KLF5 promoter activity in a concentration-dependent manner. Deletion analysis of the promoter region of the KLF5 gene revealed that the region between nucleotides-991 and -699 contains the transcriptional regulatory element stimulated by C3a. C3a induced extracellular signal-regulated kinase (ERK) phosphorylation, and C3a-increased KLF5 promoter activity was completely inhibited by the MEK inhibitor U0126. These findings suggest that C3 increases KLF5 promoter activity and gene expression via ERK signaling.
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Complemento C3/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Animales , Células Cultivadas , Masculino , RatasRESUMEN
BACKGROUND: Angiotensin II (Ang II) receptor blocker (ARB) has been reported to have protective effects on the cardiovascular system independent of blood pressure reduction. Endothelial progenitor cells (EPCs) play a significant role in neovascularization of ischemic tissue. The average lifespan of EPCs was recently reported to be shortened by oxidative stress and regulated by anti-oxidative mechanisms. It has been reported that EPCs are present in peripheral blood and have the ability to repair cardiovascular damage. We investigated the effects of an ARB, candesartan, on EPC function and cardiovascular oxidation in salt-loaded, stroke-prone, spontaneously hypertensive rats (SHR-SP) in vivo. METHODS: Salt-loaded SHR-SP were treated with candesartan (1 mg/kg/day), a diuretic (trichlormethiazide, TCM, 1.6 mg/kg/day), or an antioxidant (tempol, 5 mg/kg/day) for 2 weeks. Peripheral blood mononuclear cells (MNCs) were isolated and cultured to assay EPC colony formation and migration. Oxidative stress in EPCs was evaluated by thiobarbituric acid reactive substance (TBARS) assay. We evaluated messenger RNA (mRNA) expression of c-kit in the heart, the renin-angiotensin system (RAS) in EPC colonies, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit in cardiovascular organs. RESULTS: Candesartan and tempol, but not TCM, markedly increased EPC colony number in SHR-SP and reduced TBARS. Candesartan also significantly decreased mRNA expression of NADPH oxidase subunits in cardiovascular organs and increased cardiac c-kit mRNA expression. EPCs expressed mRNAs of renin, cathepsin D, chymase, and Ang II type 1 and type 2 receptors. CONCLUSIONS: Candesartan, an ARB, improves EPC dysfunction and increases cardiac c-kit expression through the anti-oxidative mechanism in hypertension. The local RAS induces oxidative stress and regulates the EPC functions.
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Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Antihipertensivos/farmacología , Antioxidantes/farmacología , Bencimidazoles/farmacología , Células Endoteliales/efectos de los fármacos , Hipertensión/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Células Madre/efectos de los fármacos , Tetrazoles/farmacología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Animales , Antihipertensivos/uso terapéutico , Antioxidantes/uso terapéutico , Aorta/efectos de los fármacos , Aorta/enzimología , Aorta/metabolismo , Bencimidazoles/uso terapéutico , Compuestos de Bifenilo , Presión Sanguínea/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Óxidos N-Cíclicos/farmacología , Modelos Animales de Enfermedad , Diuréticos/farmacología , Células Endoteliales/enzimología , Células Endoteliales/metabolismo , Hipertensión/metabolismo , Hipertensión/fisiopatología , Masculino , Miocardio/enzimología , Miocardio/metabolismo , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Sistema Renina-Angiotensina/efectos de los fármacos , Sistema Renina-Angiotensina/genética , Cloruro de Sodio Dietético/administración & dosificación , Marcadores de Spin , Células Madre/enzimología , Células Madre/metabolismo , Tetrazoles/uso terapéutico , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Triclormetiazida/farmacología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Complement activation contributes to tissue injury in various forms of glomerulopathy and is characterized by deposition of complement components, which accelerates the progression of chronic renal damage. We recently reported that complement 3 (C3), a critical component of the complement system, is associated with the synthetic phenotype of vascular smooth muscle cells. It is possible that C3 stimulates mesangial cells to assume the synthetic phenotype to, in turn, induce glomerular injury and sclerosis. We investigated the role of C3 in the growth and phenotype of mesangial cells. Cultured human mesangial cells (HMCs) expressed C3 mRNA and protein, and levels were increased in response to IFN-gamma and TNF-alpha. HMCs also expressed C3a receptor mRNA and protein. Exogenous C3a stimulated DNA synthesis in HMCs in a dose-dependent manner. C3a decreased expression h-caldesmon mRNA, a marker of the contractile phenotype, and increased the expression of osteopontin, matrix Gla, and collagen type1 alpha1 (collagen IV) mRNAs, which are markers of the synthetic phenotype. C3a decreased expression of alpha-smooth muscle actin in HMCs. Small interfering RNA (siRNA) targeting C3 reduced the DNA synthesis and proliferation of HMCs, increased expression of h-caldesmon mRNA, and decreased expression of osteopontin, matrix Gla, and collagen IV mRNAs in HMCs. These results indicate that C3 causes HMCs to convert to the synthetic phenotype and stimulates growth of mesangial cells, suggesting that C3 may play an important role in phenotypic regulation of mesangial cells in renal diseases.
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Complemento C3/fisiología , Células Mesangiales/citología , Animales , Biomarcadores/metabolismo , Células Cultivadas , Complemento C3/genética , Complemento C3/inmunología , Complemento C3a/genética , Complemento C3a/inmunología , Humanos , Factores Inmunológicos/inmunología , Interferón gamma/inmunología , Células Mesangiales/inmunología , Fenotipo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores de Complemento/metabolismo , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
We evaluated the effects of the angiotensin II (Ang II) receptor blocker (ARB) losartan on the formation and number of endothelial progenitor cells (EPCs) in hypertensive rats. Wistar-Kyoto (WKY) rats and stroke-prone, spontaneously hypertensive rats (SHR-SP) were salt-loaded and then treated with losartan (10 mg/kg/day), trichlormethiazide (TCM; 1.6 mg/kg/day), or tempol (1 mmol/L) for 2 weeks. Peripheral blood mononuclear cells were isolated, subjected to flow cytometric analysis to determine the number of circulating EPCs, cultured to assay EPC colony formation, and subjected to a migration chamber assay to evaluate EPC migration. Oxidative stress in EPCs was evaluated by thiobarbituric acid reactive substance (TBARS) assay. The results showed that the number, colony formation, and migration of EPCs were markedly decreased in SHR-SP compared to those in WKY rats. The TBARS scores were significantly greater in SHR-SP than in WKY rats. Losartan and TCM decreased systolic blood pressure in SHR-SP to similar levels. Losartan and tempol increased the number of circulating EPCs and colony formation, and inhibited oxidation in SHR-SP. TCM did not affect the EPC number, colony formation, or oxidation. Both losartan and TCM stimulated EPC migration. Expression of gp91(phox), p22(phox), and p47(phox) mRNA in tissues was significantly decreased by losartan but not by TCM. These results indicate that the formation and function of EPCs are impaired by oxidative stress in SHR-SP. This is the first report to show that losartan improves the proliferation and function of EPCs in hypertension, suggesting that ARBs are useful to repair hypertensive vascular injuries.
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Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Antioxidantes/farmacología , Endotelio Vascular/citología , Hipertensión/tratamiento farmacológico , Losartán/farmacología , Células Madre/efectos de los fármacos , Animales , Óxidos N-Cíclicos/farmacología , Endotelio Vascular/fisiología , Hipertensión/fisiopatología , Masculino , NADPH Oxidasas/genética , ARN Mensajero/análisis , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Cloruro de Sodio Dietético/administración & dosificación , Marcadores de Spin , Células Madre/fisiologíaRESUMEN
There is accumulating evidence that reactive oxygen species (ROS) play major roles in the initiation and progression of cardiovascular dysfunction associated with diseases such as hyperlipidemia, diabetes mellitus, hypertension, ischemic heart disease, and chronic heart failure. ROS produced by migrating inflammatory cells as well as vascular cells (endothelial cells, vascular smooth muscle cells, and adventitial fibroblasts) have distinct functional effects on each cell type. These effects include cell growth, apoptosis, migration, inflammatory gene expression and matrix regulation. ROS, through regulating vascular cell function, can play a central role in normal vascular physiology, and contribute substantially to the development of cardiovascular diseases. Excessive production of ROS is an essential mechanism underlying the pathogenesis of endothelial dysfunction and cardiovascular disease. Stem cells hold great promise for tissue repair and regenerative medicine, and endothelial progenitor cells (EPC) play a significant role in neovascularization of ischemic tissue. Recent studies have shown that cardiovascular risk factors such as hypertension, hypercholesterolemia, diabetes and cigarette smoking are inversely correlated with EPC number and function. Understanding the mechanisms, that regulate EPC function may provide new insights into the pathogenesis of vasculogenesis and may promote development of specific therapies to prevent ROS production and ultimately correct EPC dysfunction. We have demonstrated the angiotensin II receptor blockers improve EPC dysfunction through antioxidative mechanisms. In the present review, we describe our current understanding of the contributions of oxidative stress to progenitor and stem cell dysfunction in cardiovascular disease and focus on the potential mechanisms that underlie oxidative stress-induced damage of progenitor and stem cells.
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Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Estrés Oxidativo/fisiología , Células Madre/metabolismo , Células Madre/patología , Animales , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Enfermedades Cardiovasculares/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Estrés Oxidativo/efectos de los fármacos , Células Madre/efectos de los fármacosRESUMEN
Vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) show the synthetic phenotype and exaggerated growth in comparison with VSMCs from normotensive Wistar-Kyoto (WKY) rats. We investigated genes associated with the synthetic phenotype and exaggerated growth of VSMCs from SHR by microarray. Expression of 1300 transcripts was evaluated by microarray with total mRNA extracted from mid-layer aortic smooth muscle of 3-week-old SHR/Izumo and WKY/Izumo rats. mRNAs encoding sodium-dependent neurotransmitter transporter, epidermal growth factor precursor, EEF2, leptin receptor long-isoform b, clathrin assembly protein short form, and preprocomplement 3 (pre-pro-C3) were expressed only in aortic smooth muscle from SHR by microarray and by reverse-transcription polymerase chain reaction analysis. Pre-pro-C3 mRNA was detected only in cultured VSMCs from SHR. Exogenous C3 changed VSMCs to the synthetic phenotype. Antisense oligodeoxynucleotides (ODN) to C3 reduced the higher level of DNA synthesis in VSMCs from SHR. Antisense ODN to C3 increased expression of SM22alpha mRNA and decreased expression of osteopontin and matrix Gla mRNAs. It also decreased expression of growth factor mRNAs in VSMCs from SHR. In conclusion, we have shown that C3, independent of other complement molecules, has direct effects on the phenotype of VSMCs and stimulates growth of these cells. C3 is produced only by VSMCs from SHR. Therefore, C3 may be the gene underlying the synthetic phenotype and exaggerated growth of VSMCs from SHR. C3 may be a new target for the treatment of hypertension.