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BACKGROUND: Vascular smooth muscle cells (VSMCs) are highly plastic. Vessel injury induces a phenotypic transformation from differentiated to dedifferentiated VSMCs, which involves reduced expression of contractile proteins and increased production of extracellular matrix and inflammatory cytokines. This transition plays an important role in several cardiovascular diseases such as atherosclerosis, hypertension, and aortic aneurysm. TGF-ß (transforming growth factor-ß) is critical for VSMC differentiation and to counterbalance the effect of dedifferentiating factors. However, the mechanisms controlling TGF-ß activity and VSMC phenotypic regulation under in vivo conditions are poorly understood. The extracellular matrix protein TN-X (tenascin-X) has recently been shown to bind TGF-ß and to prevent it from activating its receptor. METHODS: We studied the role of TN-X in VSMCs in various murine disease models using tamoxifen-inducible SMC-specific knockout and adeno-associated virus-mediated knockdown. RESULTS: In hypertensive and high-fat diet-fed mice, after carotid artery ligation as well as in human aneurysmal aortae, expression of Tnxb, the gene encoding TN-X, was increased in VSMCs. Mice with smooth muscle cell-specific loss of TN-X (SMC-Tnxb-KO) showed increased TGF-ß signaling in VSMCs, as well as upregulated expression of VSMC differentiation marker genes during vascular remodeling compared with controls. SMC-specific TN-X deficiency decreased neointima formation after carotid artery ligation and reduced vessel wall thickening during Ang II (angiotensin II)-induced hypertension. SMC-Tnxb-KO mice lacking ApoE showed reduced atherosclerosis and Ang II-induced aneurysm formation under high-fat diet. Adeno-associated virus-mediated SMC-specific expression of short hairpin RNA against Tnxb showed similar beneficial effects. Treatment with an anti-TGF-ß antibody or additional SMC-specific loss of the TGF-ß receptor reverted the effects of SMC-specific TN-X deficiency. CONCLUSIONS: In summary, TN-X critically regulates VSMC plasticity during vascular injury by inhibiting TGF-ß signaling. Our data indicate that inhibition of vascular smooth muscle TN-X may represent a strategy to prevent and treat pathological vascular remodeling.
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Músculo Liso Vascular , Miocitos del Músculo Liso , Transducción de Señal , Tenascina , Remodelación Vascular , Animales , Humanos , Masculino , Ratones , Angiotensina II , Aneurisma de la Aorta/metabolismo , Aneurisma de la Aorta/patología , Aneurisma de la Aorta/genética , Aneurisma de la Aorta/prevención & control , Traumatismos de las Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/genética , Células Cultivadas , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Hipertensión/metabolismo , Hipertensión/patología , Hipertensión/fisiopatología , Hipertensión/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Noqueados para ApoE , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Neointima , Fenotipo , Tenascina/metabolismo , Tenascina/genética , Tenascina/deficiencia , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Hypertensive cerebrovascular remodeling involves the enlargement of vascular smooth muscle cells (VSMCs), which activates volume-regulated Cl- channels (VRCCs). The leucine-rich repeat-containing family 8 A (LRRC8A) has been shown to be the molecular identity of VRCCs. However, its role in vascular remodeling during hypertension is unclear. In this study, we used vascular smooth muscle-specific LRRC8A knockout (CKO) mice and an angiotensin II (Ang II)-induced hypertension model. The results showed that cerebrovascular remodeling during hypertension was ameliorated in CKO mice, and extracellular matrix (ECM) deposition was reduced. Based on the RNA-sequencing analysis of aortic tissues, the level of matrix metalloproteinases (MMPs), such as MMP-9 and MMP-14, were reduced in CKO mice with hypertension, which was further verified in vivo by qPCR and immunofluorescence analysis. Knockdown of LRRC8A in VSMCs inhibited the Ang II-induced upregulation of collagen I, fibronectin, and matrix metalloproteinases (MMPs), and overexpression of LRRC8A had the opposite effect. Further experiments revealed an interaction between with-no-lysine (K)-1 (WNK1), which is a "Cl--sensitive kinase", and Forkhead transcription factor O3a (FOXO3a), which is a transcription factor that regulates MMP expression. Ang II induced the phosphorylation of WNK1 and downstream FOXO3a, which then increased the expression of MMP-2 and MMP-9. This process was inhibited or potentiated when LRRC8A was knocked down or overexpressed, respectively. Overall, these results demonstrate that LRRC8A knockout in vascular smooth muscle protects against cerebrovascular remodeling during hypertension by reducing ECM deposition and inhibiting the WNK1/FOXO3a/MMP signaling pathway, demonstrating that LRRC8A is a potential therapeutic target for vascular remodeling-associated diseases such as stroke.
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Angiotensina II , Proteína Forkhead Box O3 , Hipertensión , Ratones Noqueados , Músculo Liso Vascular , Transducción de Señal , Remodelación Vascular , Proteína Quinasa Deficiente en Lisina WNK 1 , Animales , Músculo Liso Vascular/metabolismo , Proteína Forkhead Box O3/metabolismo , Proteína Forkhead Box O3/genética , Ratones , Proteína Quinasa Deficiente en Lisina WNK 1/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1/genética , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Hipertensión/genética , Masculino , Metaloproteinasas de la Matriz/metabolismo , Ratones Endogámicos C57BL , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Células CultivadasRESUMEN
BACKGROUND: The prognostic value of traditional clinical indicators for locally recurrent nasopharyngeal carcinoma is limited because of their inability to reflect intratumor heterogeneity. We aimed to develop a radiomic signature to reveal tumor immune heterogeneity and predict survival in locally recurrent nasopharyngeal carcinoma. METHODS: This multicenter, retrospective study included 921 patients with locally recurrent nasopharyngeal carcinoma. A machine learning signature and nomogram based on pretreatment magnetic resonance imaging features were developed for predicting overall survival in a training cohort and validated in 2 independent cohorts. A clinical nomogram and an integrated nomogram were constructed for comparison. Nomogram performance was evaluated by concordance index and receiver operating characteristic curve analysis. Accordingly, patients were classified into risk groups. The biological characteristics and immune infiltration of the signature were explored by RNA-sequencing analysis. RESULTS: The machine learning signature and nomogram demonstrated comparable prognostic ability to a clinical nomogram, achieving concordance indexes of 0.729, 0.718, and 0.731 in the training, internal, and external validation cohorts, respectively. Integration of the signature and clinical variables statistically improved the predictive performance. The proposed signature effectively distinguished patients between risk groups with statistically distinct overall survival rates. Subgroup analysis indicated the recommendation of local salvage treatments for low-risk patients. Exploratory RNA-sequencing analysis revealed differences in interferon response and lymphocyte infiltration between risk groups. CONCLUSIONS: A magnetic resonance imaging-based radiomic signature predicted overall survival more accurately. The proposed signature associated with tumor immune heterogeneity may serve as a valuable tool to facilitate prognostic stratification and guide individualized management for locally recurrent nasopharyngeal carcinoma patients.
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Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Recurrencia Local de Neoplasia , Nomogramas , Radiómica , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Aprendizaje Automático , Imagen por Resonancia Magnética , Carcinoma Nasofaríngeo/mortalidad , Carcinoma Nasofaríngeo/inmunología , Carcinoma Nasofaríngeo/diagnóstico por imagen , Carcinoma Nasofaríngeo/patología , Neoplasias Nasofaríngeas/mortalidad , Neoplasias Nasofaríngeas/diagnóstico por imagen , Neoplasias Nasofaríngeas/inmunología , Neoplasias Nasofaríngeas/patología , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
BACKGROUND: Radiotherapy-related toxicities of nasopharyngeal carcinoma (NPC) caused by a standard dose of 70 Gy remain a critical issue. Therefore, we assessed whether a radiotherapy dose of 60 Gy was non-inferior to the standard dose in patients with low-risk stage III NPC with a favourable response to induction chemotherapy (IC). PATIENTS AND METHODS: We did a single-arm, single-centre, phase II clinical trial in China. Patients with low-risk (Epstein-Barr virus [EBV] DNA level <4000 copies/ml) stage III NPC were treated with two cycles IC. Patients with complete/partial response and undetectable EBV DNA level were assigned 60 Gy intensity-modulated radiotherapy concurrently with three cycles of cisplatin. The primary end-point was 2-year progression-free survival (PFS). This trial is registered with ClinicalTrials.gov, number NCT03668730. RESULTS: One patient quit because of withdrawal of informed consent after IC. In total, 215 patients completed two cycles of IC, after which 116 (54.0%) and 99 (46.0%) patients were assigned 60 and 70 Gy radiotherapy, respectively. For 215 patients, the 2-year PFS was 90.7% (95% CI, 86.8%-94.6%) with a median follow-up of 43.9 months (interquartile range [IQR], 39.8-46.2). For patients treated with 60 Gy radiotherapy, the 2-year PFS rate was 94.8% (95%CI 90.7%-98.9%) with a median follow-up of 43.9 months (IQR 40.2-46.2). The most common late toxicity was grade 1-2 dry mouth (incidence rate: 54.3%). No grade 3+ long-term adverse event was observed, and most quality-of-life items, domains, and symptom scores returned to baseline by 6 months. CONCLUSION: Reduced-dose radiation (60 Gy) is associated with favourable survival outcomes and limited treatment-related toxicities in patients with low-risk stage III NPC sensitive to IC.
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Infecciones por Virus de Epstein-Barr , Neoplasias Nasofaríngeas , Radioterapia de Intensidad Modulada , Humanos , Carcinoma Nasofaríngeo/radioterapia , Carcinoma Nasofaríngeo/tratamiento farmacológico , Herpesvirus Humano 4/genética , Neoplasias Nasofaríngeas/tratamiento farmacológico , Infecciones por Virus de Epstein-Barr/complicaciones , Supervivencia sin Enfermedad , Quimioradioterapia/efectos adversos , Radioterapia de Intensidad Modulada/efectos adversos , ADN ViralRESUMEN
Background: Previous studies demonstrated that induction chemotherapy (IC) followed by de-escalated chemoradiotherapy adapted to tumor response was effective in treating childhood nasopharyngeal carcinoma (NPC), but the toxicity profile of this treatment strategy, and whether childhood patients with advanced stages can obtain enough benefits from it requires further investigation. Methods: We conducted a single-center phase II trial (NCT03020329). All participants received 3 cycles of paclitaxel liposome, cisplatin and 5-fluorouracil (TPF)-based IC. Patients who showed complete or partial response received de-escalated radiotherapy of 60 Gy with 3 cycles of concurrent cisplatin, and those who showed stable or progressive disease received standard-dose radiotherapy of 70 Gy with concurrent cisplatin. The primary endpoint was the complete response (CR) rate at the end of concurrent chemoradiotherapy (CCRT). Findings: From November 2016 to March 2021, 44 patients were recruited in the cohort. The CR rate was 80% (35/44, 95% CI, 65-90) of the whole cohort. All patients achieved CR 3 months after CCRT. By the last follow-up, the 3-year progression-free survival and overall survival were 91% (95% CI, 82-99) and 100% respectively. Dry mouth was the most common late toxicity, with an incidence of 41% (18/44), followed by skin fibrosis and hearing impairment. No patient suffered from severe late toxicity and growth retardation. Interpretation: Our results proved the efficacy and safety of TPF regimen followed by de-escalated radiotherapy with concurrent cisplatin in treating stage IVa-b childhood NPC patients. Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.
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Immunotherapy combined with antiangiogenic targeted therapy has improved the treatment of certain solid tumors, but effective regimens remain elusive for refractory recurrent/metastatic nasopharyngeal carcinoma (RM-NPC). We conducted a phase 2 trial to evaluate the safety and activity of camrelizumab plus apatinib in platinum-resistant (cohort 1, NCT04547088) and PD-1 inhibitor resistant NPC (cohort 2, NCT04548271). Here we report on the primary outcome of objective response rate (ORR) and secondary endpoints of safety, duration of response, disease control rate, progression-free survival, and overall survival. The primary endpoint of ORR was met for cohort 1 (65%, 95% CI, 49.6-80.4, n = 40) and cohort 2 (34.3%; 95% CI, 17.0-51.8, n = 32). Grade ≥ 3 treatment-related adverse events (TRAE) were reported in 47 (65.3%) of 72 patients. Results of our predefined exploratory investigation of predictive biomarkers show: B cell markers are the most differentially expressed genes in the tumors of responders versus non-responders in cohort 1 and that tertiary lymphoid structure is associated with higher ORR; Angiogenesis gene expression signatures are strongly associated with ORR in cohort 2. Camrelizumab plus apatinib combination effectiveness is associated with high expression of PD-L1, VEGF Receptor 2 and B-cell-related genes signatures. Camrelizumab plus apatinib shows promising efficacy with a measurable safety profile in RM-NPC patients.
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Inhibidores de Puntos de Control Inmunológico , Neoplasias Nasofaríngeas , Humanos , Carcinoma Nasofaríngeo/tratamiento farmacológico , Platino (Metal) , Neoplasias Nasofaríngeas/tratamiento farmacológico , Neoplasias Nasofaríngeas/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéuticoRESUMEN
OBJECTIVES: To evaluate whether MRI-based T stage (TMRI), [18F]FDG PET/CT-based N (NPET/CT), and M stage (MPET/CT) are superior in NPC patients' prognostic stratification based on long-term survival evidences, and whether TNM staging method involving TMRI + NPET/CT + MPET/CT could improve NPC patients' prognostic stratification. METHODS: From April 2007 to December 2013, 1013 consecutive untreated NPC patients with complete imaging data were enrolled. All patients' initial stages were repeated based on (1) the NCCN guideline recommended "TMRI + NMRI + MPET/CT" ("MMP") staging method; (2) the traditional "TMRI + NMRI + Mconventional work-up (CWU)" ("MMC") staging method; (3) the single-step "TPET/CT + NPET/CT + MPET/CT" ("PPP") staging method; or (4) the "TMRI + NPET/CT + MPET/CT" ("MPP") staging method recommended in present research. Survival curve, ROC curve, and net reclassification improvement (NRI) analysis were used to evaluate the prognosis predicting ability of different staging methods. RESULTS: [18F]FDG PET/CT performed worse on T stage (NRI = - 0.174, p < 0.001) but better on N (NRI = 0.135, p = 0.004) and M stage (NRI = 0.126, p = 0.001). The patients whose N stage upgraded by [18F]FDG PET/CT had worse survival (p = 0.011). The "TMRI + NPET/CT + MPET/CT" ("MPP") method performed better on survival prediction when compared with "MMP" (NRI = 0.079, p = 0.007), "MMC" (NRI = 0.190, p < 0.001), or "PPP" method (NRI = 0.107, p < 0.001). The "TMRI + NPET/CT + MPET/CT" ("MPP") method could reclassify patients' TNM stage to a more appropriate stage. The improvement is significant in patients with more than 2.5-years follow-up according to the time-dependent NRI values. CONCLUSIONS: The MRI is superior to [18F]FDG PET/CT in T stage, and [18F]FDG PET/CT is superior to CWU in N/M stage. The "TMRI + NPET/CT + MPET/CT" ("MPP") staging method could significantly improve NPC patients' long-term prognostic stratification. CLINICAL RELEVANCE STATEMENT: The present research provided long-term follow-up evidence for benefits of MRI and [18F]FDG PET/CT in TNM staging for nasopharyngeal carcinoma, and proposes a new imaging procedure for TNM staging incorporating MRI-based T stage and [18F]FDG PET/CT-based N and M stage, which significantly improves long-term prognostic stratification for patients with NPC. KEY POINTS: ⢠The long-term follow-up evidence of a large-scale cohort was provided to evaluate the advantages of MRI, [18F]FDG PET/CT, and CWU in the TNM staging of nasopharyngeal carcinoma. ⢠A new imaging procedure for TNM stage of nasopharyngeal carcinoma was proposed.
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Neoplasias Nasofaríngeas , Tomografía Computarizada por Tomografía de Emisión de Positrones , Humanos , Carcinoma Nasofaríngeo/diagnóstico por imagen , Carcinoma Nasofaríngeo/patología , Pronóstico , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Fluorodesoxiglucosa F18 , Radiofármacos , Tomografía de Emisión de Positrones/métodos , Estadificación de Neoplasias , Imagen por Resonancia Magnética , Neoplasias Nasofaríngeas/patologíaRESUMEN
Platelet hyperactivity is essential for thrombus formation in coronary artery diseases (CAD). Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) in patients with cystic fibrosis elevates intracellular Cl- levels ([Cl-]i) and enhanced platelet hyperactivity. In this study, we explored whether alteration of [Cl-]i has a pathological role in regulating platelet hyperactivity and arterial thrombosis formation. CFTR expression was significantly decreased, while [Cl-]i was increased in platelets from CAD patients. In a FeCl3-induced mouse mesenteric arteriole thrombosis model, platelet-specific Cftr-knockout and/or pre-administration of ion channel inhibitor CFTRinh-172 increased platelet [Cl-]i, which accelerated thrombus formation, enhanced platelet aggregation and ATP release, and increased P2Y12 and PAR4 expression in platelets. Conversely, Cftr-overexpressing platelets resulted in subnormal [Cl-]i, thereby decreasing thrombosis formation. Our results showed that clamping [Cl-]i at high levels or Cftr deficiency-induced [Cl-]i increasement dramatically augmented phosphorylation (Ser422) of serum and glucocorticoid-regulated kinase (SGK1), subsequently upregulated P2Y12 and PAR4 expression via NF-κB signaling. Constitutively active mutant S422D SGK1 markedly increased P2Y12 and PAR4 expression. The specific SGK1 inhibitor GSK-650394 decreased platelet aggregation in wildtype and platelet-specific Cftr knockout mice, and platelet SGK1 phosphorylation was observed in line with increased [Cl-]i and decreased CFTR expression in CAD patients. Co-transfection of S422D SGK1 and adenovirus-induced CFTR overexpression in MEG-01 cells restored platelet activation signaling cascade. Our results suggest that [Cl-]i is a novel positive regulator of platelet activation and arterial thrombus formation via the activation of a [Cl-]i-sensitive SGK1 signaling pathway. Therefore, [Cl-]i in platelets is a novel potential biomarker for platelet hyperactivity, and CFTR may be a potential therapeutic target for platelet activation in CAD.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística , Proteínas Inmediatas-Precoces , Trombosis , Adenosina Trifosfato/metabolismo , Animales , Plaquetas/metabolismo , Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Trombosis/metabolismoRESUMEN
BACKGROUND: Low serum chloride (Cl-) level is considered an independent predictor of cardiovascular mortality associated with chronic hypertension. However, the underlying mechanisms are unknown. ClC-5, a member of the Cl- channel family, is sensitive to changes in intracellular and extracellular Cl- concentration and conducts outwardly rectifying Cl- currents. The aims of this study were to determine if ClC-5 is regulated by low extracellular Cl-, clarify its putative roles in hypertension-induced cerebrovascular remodeling, and elucidate the associated underlying mechanisms. METHODS: Whole-cell patch technique, intracellular Cl- concentration measurements, flow cytometry, Western blot, Clcn5 knockdown (Clcn5-/y), and adenovirus-mediated ClC-5 overexpression mice, 2-kidney, 2-clip, and angiotensin II infusion-induced hypertensive models were used. RESULTS: We found that low extracellular Cl- evoked a ClC-5-dependent Cl- current that was abolished by ClC-5 depletion in basilar artery smooth muscle cells (BASMCs). ClC-5 was upregulated in the arterial tissues of rats and patients with hypertension. Low Cl--induced current and ClC-5 protein expression positively correlated with basilar artery remodeling during hypertension. ClC-5 knockdown ameliorated hypertension-induced cerebrovascular remodeling and smooth muscle cell proliferation, whereas ClC-5 overexpression mice exhibited the opposite phenotype. ClC-5-dependent Cl- efflux induced by low extracellular Cl- activated WNK1 (lysine-deficient protein kinase 1) which, in turn, activated AKT (protein kinase B), and culminated in BASMC proliferation and vascular remodeling. CONCLUSIONS: ClC-5 mediates low extracellular Cl-induced Cl- currents in BASMCs and regulates hypertension-induced cerebrovascular remodeling by promoting BASMC proliferation via the WNK1/AKT signaling pathway.
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Hipertensión , Proteínas Proto-Oncogénicas c-akt , Animales , Proliferación Celular , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Humanos , Ratones , Miocitos del Músculo Liso/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , RatasRESUMEN
OBJECTIVES: Recent studies revealed LRRC8A to be an essential component of volume-regulated anion channel (VRAC), which regulates cellular volume homeostasis. However, evidence for the contribution of LRRC8A-dependent VRAC activity in vascular smooth muscle cells (VSMCs) is still lacking, and the relevant functional role of LRRC8A in VSMCs remains unknown. The primary goal of this study was to elucidate the role of LRRC8A in VRAC activity in VSMCs and the functional role of LRRC8A in cerebrovascular remodeling during hypertension. MATERIALS AND METHODS: siRNA-mediated knockdown and adenovirus-mediated overexpression of LRRC8A were used to elucidate the electrophysiological properties of LRRC8A in basilar smooth muscle cells (BASMCs). A smooth muscle-specific overexpressing transgenic mouse model was used to investigate the functional role of LRRC8A in cerebrovascular remodeling. RESULTS: LRRC8A is essential for volume-regulated chloride current (ICl, Vol ) in BASMCs. Overexpression of LRRC8A induced a voltage-dependent Cl- current independently of hypotonic stimulation. LRRC8A regulated BASMCs proliferation through activation of WNK1/PI3K-p85/AKT axis. Smooth muscle-specific upregulation of LRRC8A aggravated Angiotensin II-induced cerebrovascular remodeling in mice. CONCLUSIONS: LRRC8A is an essential component of VRAC and is required for cell volume homeostasis during osmotic challenge in BASMCs. Smooth muscle specific overexpression of LRRC8A increases BASMCs proliferation and substantially aggravates basilar artery remodeling, revealing a potential therapeutic target for vascular remodeling in hypertension.
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Arteria Basilar/metabolismo , Circulación Cerebrovascular , Hipertensión/metabolismo , Proteínas de la Membrana/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Remodelación Vascular , Animales , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
AIMS: Our previous study demonstrated that Ca2+ influx through the Orai1 store-operated Ca2+ channel in macrophages contributes to foam cell formation and atherosclerosis via the calcineurin-ASK1 pathway, not the classical calcineurin-nuclear factor of activated T-cell (NFAT) pathway. Moreover, up-regulation of NFATc3 in macrophages inhibits foam cell formation, suggesting that macrophage NFATc3 is a negative regulator of atherogenesis. Hence, this study investigated the precise role of macrophage NFATc3 in atherogenesis. METHODS AND RESULTS: Macrophage-specific NFATc3 knockout mice were generated to determine the effect of NFATc3 on atherosclerosis in a mouse model of adeno-associated virus-mutant PCSK9-induced atherosclerosis. NFATc3 expression was decreased in macrophages within human and mouse atherosclerotic lesions. Moreover, NFATc3 levels in peripheral blood mononuclear cells from atherosclerotic patients were negatively associated with plaque instability. Furthermore, macrophage-specific ablation of NFATc3 in mice led to the atherosclerotic plaque formation, whereas macrophage-specific NFATc3 transgenic mice exhibited the opposite phenotype. NFATc3 deficiency in macrophages promoted foam cell formation by potentiating SR-A- and CD36-meditated lipid uptake. NFATc3 directly targeted and transcriptionally up-regulated miR-204 levels. Mature miR-204-5p suppressed SR-A expression via canonical regulation. Unexpectedly, miR-204-3p localized in the nucleus and inhibited CD36 transcription. Restoration of miR-204 abolished the proatherogenic phenotype observed in the macrophage-specific NFATc3 knockout mice, and blockade of miR-204 function reversed the beneficial effects of NFATc3 in macrophages. CONCLUSION: Macrophage NFATc3 up-regulates miR-204 to reduce SR-A and CD36 levels, thereby preventing foam cell formation and atherosclerosis, indicating that the NFATc3/miR-204 axis may be a potential therapeutic target against atherosclerosis.
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Aterosclerosis , MicroARNs , Animales , Aterosclerosis/genética , Células Espumosas , Humanos , Leucocitos Mononucleares , Ratones , MicroARNs/genética , Factores de Transcripción NFATC/genética , Proproteína Convertasa 9RESUMEN
The inflammatory response of endothelial cells accelerates various vascular diseases. MicroRNAs (miRNAs) participate in diverse cellular processes during inflammation. In the present study, we found that miR-302a is an effective suppressor of vascular inflammation in endothelial cells. It was revealed that miR-302a exhibited a lower level in a lipopolysaccharide (LPS)-induced mouse model and in patients with vascular inflammatory disease. Genetic haploinsufficiency of miR-302 aggravated the LPS-induced vascular inflammatory response in mice, and overexpression of miR-302a attenuated vascular inflammation in mice. Furthermore, overexpression of miR-302a inhibited the synthesis and secretion of adhesion factors in endothelial cells, and suppressed the adhesion of monocytes to endothelium. In the study of molecular mechanism, we found that miR-302a relieved vascular inflammation mainly by regulating the nuclear factor kappa-B (NF-κB) pathway in endothelial cells. The results showed that interleukin-1 receptor-associated kinase4 (IRAK4) and zinc finger protein 91 (ZFP91) were the binding targets of miR-302a. MiR-302a prevented the nuclear translocation of NF-κB by inhibiting phosphorylation of IκB kinase complex ß (IKKß) and inhibitors of κBα (IκBα) via targeting IRAK4. In addition, miR-302a downregulated the expression of NF-κB by directly binding with ZFP91. These findings indicate that miR-302a negatively regulates inflammatory responses in the endothelium via the NF-κB pathway and it may be a novel target for relieving vascular inflammation.
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Hypotonic stimulus enlarges cell volume and increased cell proliferation with the exact mechanisms unknown. Glucocorticoid-induced kinase-1 (SGK1) is a serine/threonine kinase that can be regulated by osmotic pressure. We have revealed that SGK1 was activated by hypotonic solution-induced lowering of intracellular Cl- concentration. Therefore, we further examined whether SGK1 mediated hypotonic solution-induced proliferation and the internal mechanisms in basilar smooth muscle cells (BASMCs). In the present study, BrdU incorporation assay, flow cytometry, western blotting were performed to evaluate cell viability, cell cycle transition, and the expression of cell cycle regulators and other related proteins. We found that silence of SGK1 largely blunted hypotonic challenge-induced increase in cell viability and cell cycle transition from G0/G1 phase to S phase, whereas overexpression of SGK1 showed the opposite effects. The effect of SGK1 on proliferation was related to the upregulation of cyclin D1 and cyclin E1, and the downregulation of p27 and p21, which is mediated by the interaction between SGK1 and cAMP responsive element-binding protein (CREB). Moreover, we overexpressed ClC-3 Cl- channel to further verify the role of SGK1 in low Cl- environment-induced proliferation. The results revealed that overexpression of ClC-3 further enhanced hypotonic solution-induced cell viability, cell cycle transition, and CREB activation, which were alleviated or potentiated by silencing or overexpression of SGK1. In summary, this study provides compelling evidences that SGK1, as a Cl--sensitive kinase, is a critical link between low osmotic pressure and proliferation in BASMCs, and shed a new light on the treatment of proliferation-associated cardiovascular diseases.
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Proliferación Celular/efectos de los fármacos , Canales de Cloruro/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Soluciones Hipotónicas/farmacología , Proteínas Inmediatas-Precoces/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Arteria Basilar/efectos de los fármacos , Arteria Basilar/enzimología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Canales de Cloruro/genética , Proteínas Inmediatas-Precoces/genética , Masculino , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Presión Osmótica , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Ratas Sprague-Dawley , Transducción de SeñalRESUMEN
OBJECTIVES: The value of using PET/CT for staging of stage I-II NPC remains unclear. Hence, we aimed to investigate the survival benefit of PET/CT for staging of early-stage NPC before radical therapy. METHODS: A total of 1003 patients with pathologically confirmed NPC of stages I-II were consecutively enrolled. Among them, 218 patients underwent both PET/CT and conventional workup ([CWU], head-and-neck MRI, chest radiograph, liver ultrasound, bone scintigraphy) before treatment. The remaining 785 patients only underwent CWU. The standard of truth (SOT) for lymph node metastasis was defined by the change of size according to follow-up MRI. The diagnostic efficacies were compared in 218 patients who underwent both PET/CT and CWU. After covariate adjustment using propensity scoring, a cohort of 872 patients (218 with and 654 without pre-treatment PET/CT) was included. The primary outcome was overall survival based on intention to treat. RESULTS: Retropharyngeal lymph nodes were metastatic based on follow-up MRI in 79 cases. PET/CT was significantly less sensitive than MRI in detecting retropharyngeal lymph node lesions (72.2% [62.3-82.1] vs. 91.1% [84.8-97.4], p = 0.004). Neck lymph nodes were metastatic in 89 cases and PET/CT was more sensitive than MRI (96.6% [92.8-100.0] vs. 76.4% [67.6-85.2], p < 0.001). In the survival analyses, there was no association between pre-treatment PET/CT use and improved overall survival, progression-free survival, local relapse-free survival, regional relapse-free survival, and distant metastasis-free survival. CONCLUSIONS: This study showed PET/CT is of little value for staging of stage I-II NPC patients at initial imaging. KEY POINTS: ⢠PET/CT was more sensitive than MRI in detecting neck lymph node lesions whereas it was significantly less sensitive than MRI in detecting retropharyngeal lymph node lesions. ⢠No association existed between pre-treatment PET/CT use and improved survival in stage I-II NPC patients.
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Neoplasias Nasofaríngeas , Tomografía Computarizada por Tomografía de Emisión de Positrones , Estudios de Casos y Controles , Fluorodesoxiglucosa F18 , Humanos , Ganglios Linfáticos/patología , Carcinoma Nasofaríngeo/diagnóstico por imagen , Neoplasias Nasofaríngeas/diagnóstico por imagen , Neoplasias Nasofaríngeas/patología , Recurrencia Local de Neoplasia , Estadificación de Neoplasias , Tomografía de Emisión de Positrones , Radiofármacos , Tomografía Computarizada por Rayos XRESUMEN
PURPOSE: This study aimed to establish an effective nomogram to predict primary distant metastasis (DM) in patients with nasopharyngeal carcinoma (NPC) to guide the application of PET/CT. METHODS: In total, 3591 patients with pathologically confirmed NPC were consecutively enrolled. The nomogram was constructed based on 1922 patients treated between 2007 and 2014. Multivariate logistical regression was applied to identify the independent risk factors of DM. The predictive value of the nomogram was evaluated using the concordance index (C-index), calibration curve, probability density functions (PDFs), and clinical utility curve (CUC). The results were validated in 1669 patients enrolled from 2015 to 2016. Net reclassification improvement (NRI) was applied to compare performances of the nomogram with other clinical factors. The best cut-off value of the nomogram chosen for clinical application was analyzed. RESULTS: A total of 355 patients showed primary DM among 3591 patients, yielding an incidence rate of 9.9%. Sex, N stage, EBV DNA level, lactate dehydrogenase level, and hemoglobin level were independent predictive factors for primary DM. C-indices in the training and validation cohort were 0.796 (95% CI, 0.76-0.83) and 0.779 (95% CI, 0.74-0.81), respectively. The NRI indices demonstrated that this model had better predictive performance than plasma EBV DNA level and N stage. We advocate for a threshold probability of 3.5% for guiding the application of PET/CT depending on the clinical utility analyses. CONCLUSION: This nomogram is a useful tool to predict primary DM of NPC and guide the clinical application of PET/CT individually at the initial staging.
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Neoplasias Nasofaríngeas , Nomogramas , Fluorodesoxiglucosa F18 , Humanos , Carcinoma Nasofaríngeo/diagnóstico por imagen , Neoplasias Nasofaríngeas/diagnóstico por imagen , Estadificación de Neoplasias , Tomografía Computarizada por Tomografía de Emisión de Positrones , PronósticoRESUMEN
Chloride (Cl-) homeostasis is of great significance in cardiovascular system. Serum Cl- level is inversely associated with the mortality of patients with heart failure. Considering the importance of angiogenesis in the progress of heart failure, this study aims to investigate whether and how reduced intracellular Cl- concentration ([Cl-]i) affects angiogenesis. Human umbilical endothelial cells (HUVECs) were treated with normal Cl- medium or low Cl- medium. We showed that reduction of [Cl-]i (from 33.2 to 16.18 mM) inhibited HUVEC proliferation, migration, cytoskeleton reorganization, tube formation, and subsequently suppressed angiogenesis under basal condition, and VEGF stimulation or hypoxia treatment. Moreover, VEGF-induced NADPH-mediated reactive oxygen species (ROS) generation and VEGFR2 axis activation were markedly attenuated in low Cl- medium. We revealed that lowering [Cl-]i inhibited the expression of the membrane-bound catalytic subunits of NADPH, i.e., p22phox and Nox2, and blunted the translocation of cytosolic regulatory subunits p47phox and p67phox, thereby restricting NADPH oxidase complex formation and activation. Furthermore, reduced [Cl-]i enhanced ROS-associated protein tyrosine phosphatase 1B (PTP1B) activity and increased the interaction of VEGFR2 and PTP1B. Pharmacological inhibition of PTP1B reversed the effect of lowering [Cl-]i on VEGFR2 phosphorylation and angiogenesis. In mouse hind limb ischemia model, blockade of Cl- efflux using Cl- channel inhibitors DIDS or DCPIB (10 mg/kg, i.m., every other day for 2 weeks) significantly enhanced blood flow recovery and new capillaries formation. In conclusion, decrease of [Cl-]i suppresses angiogenesis via inhibiting oxidase stress-mediated VEGFR2 signaling activation by preventing NADPH oxidase complex formation and promoting VEGFR2/PTP1B association, suggesting that modulation of [Cl-]i may be a novel therapeutic avenue for the treatment of angiogenic dysfunction-associated diseases.
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Cloruros/metabolismo , Neovascularización Fisiológica/fisiología , Estrés Oxidativo/fisiología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Citoesqueleto de Actina/fisiología , Animales , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Miembro Posterior/irrigación sanguínea , Células Endoteliales de la Vena Umbilical Humana , Humanos , Isquemia/metabolismo , Ratones Endogámicos C57BL , NADPH Oxidasa 2/metabolismo , NADPH Oxidasas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is the most prevalent form of chronic liver disease, and the mechanisms underpinning its pathogenesis have not been completely established. Transmembrane member 16A (TMEM16A), a component of the Ca2+-activated chloride channel (CaCC), has recently been implicated in metabolic events. Herein, TMEM16A is shown to be responsible for CaCC activation in hepatocytes and is increased in liver tissues of mice and patients with NAFLD. Hepatocyte-specific ablation of TMEM16A in mice ameliorates high-fat diet-induced obesity, hepatic glucose metabolic disorder, steatosis, insulin resistance, and inflammation. In contrast, hepatocyte-specific TMEM16A transgenic mice exhibit the opposite phenotype. Mechanistically, hepatocyte TMEM16A interacts with vesicle-associated membrane protein 3 (VAMP3) to induce its degradation, suppressing the formation of the VAMP3/syntaxin 4 and VAMP3/synaptosome-associated protein 23 complexes. This leads to the impairment of hepatic glucose transporter 2 (GLUT2) translocation and glucose uptake. Notably, VAMP3 overexpression restrains the functions of hepatocyte TMEM16A in blocking GLUT2 translocation and promoting lipid deposition, insulin resistance, and inflammation. In contrast, VAMP3 knockdown reverses the beneficial effects of TMEM16A downregulation. This study demonstrates a role for TMEM16A in NAFLD and suggests that inhibition of hepatic TMEM16A or disruption of TMEM16A/VAMP3 interaction may provide a new potential therapeutic strategy for NAFLD.
RESUMEN
Rationale: Transmembrane member 16A (TMEM16A) is a component of calcium-activated chloride channels that regulate vascular smooth muscle cell (SMC) proliferation and remodeling. Autophagy, a highly conserved cellular catabolic process in eukaryotes, exerts important physiological functions in vascular SMCs. In the current study, we investigated the relationship between TMEM16A and autophagy during vascular remodeling. Methods: We generated a transgenic mouse that overexpresses TMEM16A specifically in vascular SMCs to verify the role of TMEM16A in vascular remodeling. Techniques employed included immunofluorescence, electron microscopy, co-immunoprecipitation, and Western blotting. Results: Autophagy was activated in aortas from angiotensin II (AngII)-induced hypertensive mice with decreased TMEM16A expression. The numbers of light chain 3B (LC3B)-positive puncta in aortas correlated with the medial cross-sectional aorta areas and TMEM16A expression during hypertension. SMC-specific TMEM16A overexpression markedly inhibited AngII-induced autophagy in mouse aortas. Moreover, in mouse aortic SMCs (MASMCs), AngII-induced autophagosome formation and autophagic flux were blocked by TMEM16A upregulation and were promoted by TMEM16A knockdown. The effect of TMEM16A on autophagy was independent of the mTOR pathway, but was associated with reduced kinase activity of the vacuolar protein sorting 34 (VPS34) enzyme. Overexpression of VPS34 attenuated the effect of TMEM16A overexpression on MASMC proliferation, while the effect of TMEM16A downregulation was abrogated by a VPS34 inhibitor. Further, co-immunoprecipitation assays revealed that TMEM16A interacts with p62. TMEM16A overexpression inhibited AngII-induced p62-Bcl-2 binding and enhanced Bcl-2-Beclin-1 interactions, leading to suppression of Beclin-1/VPS34 complex formation. However, TMEM16A downregulation showed the opposite effects. Conclusion: TMEM16A regulates the four-way interaction between p62, Bcl-2, Beclin-1, and VPS34, and coordinately prevents vascular autophagy and remodeling.
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Anoctamina-1/fisiología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Remodelación Vascular , Animales , Autofagia , Células Cultivadas , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factor de Transcripción TFIIH/metabolismoRESUMEN
Serum- and glucocorticoid-inducible kinease-1 (SGK1) is a serine/threonine kinase regulated by hypotonic stimuli, which is involved in regulation of cell cycle and apoptosis. Our previous study shows that activation of volume-regulated Cl- channels (VRCCs) protects rat basilar artery smooth muscle cells (BASMCs) against hydrogen peroxide (H2O2)-induced apoptosis. In the present study, we investigated whether SGK1 was involved in the protective effect of VRCCs in BASMCs. We showed that hypotonic challenge significantly reduced H2O2-induced apoptosis, and increased SGK1 phosphorylation, but did not affect SGK1 protein expression. The protective effect of hypotonic challenge against H2O2-induced apoptosis was mediated through inhibiting mitochondria-dependent apoptotic pathway, evidenced by increased Bcl-2/Bax ratio, stabilizing mitochondrial membrane potential (MMP), decreased cytochrome c release from the mitochondria to the cytoplasm, and inhibition of the activation of caspase-9 and caspase-3. These protective effects of hypotonic challenge against H2O2-induced apoptosis was diminished and enhanced, respectively, by SGK1 knockdown and overexpression. We further revealed that SGK1 activation significantly increased forkhead box O3a (FOXO3a) phosphorylation, and then inhibited the translocation of FOXO3a into nucleus and the subsequent expression of Bcl-2 interacting mediator of cell death (Bim). In conclusion, SGK1 mediates the protective effect of VRCCs against H2O2-induced apoptosis in BASMCs via inhibiting FOXO3a/Bim signaling pathway. Our results provide compelling evidences that SGK1 is a critical link between VRCCs and apoptosis, and shed a new light on the treatment of vascular apoptosis-associated diseases, such as vascular remodeling, angiogenesis, and atherosclerosis.
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Apoptosis/efectos de los fármacos , Canales de Cloruro/fisiología , Peróxido de Hidrógeno/farmacología , Proteínas Inmediatas-Precoces/fisiología , Presión Osmótica/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Transducción de Señal/fisiología , Animales , Arteria Basilar/citología , Proteína 11 Similar a Bcl2/metabolismo , Regulación hacia Abajo , Proteína Forkhead Box O3/metabolismo , Masculino , Miocitos del Músculo Liso , Ratas Sprague-DawleyRESUMEN
TMEM16A Ca2+-activated chloride channel (CaCC) plays an essential role in vascular homeostasis. In this study we investigated the molecular mechanisms underlying downregulation of TMEM16A CaCC activity during hypertension. In cultured basilar artery smooth muscle cells (BASMCs) isolated from 2k2c renohypertesive rats, treatment with angiotensin II (0.125-1 µM) dose-dependently increased endophilin A2 levels and decreased TMEM16A expression. Similar phenomenon was observed in basilar artery isolated from 2k2c rats. We then used whole-cell recording to examine whether endophilin A2 could regulate TMEM16A CaCC activity in BASMCs and found that knockdown of endophilin A2 significantly enhanced CaCC activity, whereas overexpression of endophilin A2 produced the opposite effect. Overexpression of endophilin A2 did not affect the TMEM16A mRNA level, but markedly decreased TMEM16A protein level in BASMCs by inducing ubiquitination and autophagy of TMEM16A. Ubiquitin-binding receptor p62 (SQSTM1) could bind to ubiquitinated TMEM16A and resulted in a process of TMEM16A proteolysis in autophagosome/lysosome. These data provide new insights into the regulation of TMEM16A CaCC activity by endophilin A2 in BASMCs, which partly explains the mechanism of angiotensin-II-induced TMEM16A inhibition during hypertension-induced vascular remodeling.