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Piezo1 belongs to mechano-activatable cation channels serving as biological force sensors. However, the molecular events downstream of Piezo1 activation remain unclear. In this study, we used biosensors based on fluorescence resonance energy transfer (FRET) to investigate the dynamic modes of Piezo1-mediated signaling and revealed a bimodal pattern of Piezo1-induced intracellular calcium signaling. Laser-induced shockwaves (LIS) and its associated shear stress can mechanically activate Piezo1 to induce transient intracellular calcium (Ca[i] ) elevation, accompanied by an increase in FAK activity. Interestingly, multiple pulses of shockwave stimulation caused a more sustained calcium increase and a decrease in FAK activity. Similarly, tuning the degree of Piezo1 activation by titrating either the dosage of Piezo1 ligand Yoda1 or the expression level of Piezo1 produced a similar bimodal pattern of FAK responses. Further investigations revealed that SHP2 serves as an intermediate regulator mediating this bimodal pattern in Piezo1 sensing and signaling. These results suggest that the degrees of Piezo1 activation induced by both mechanical LIS and chemical ligand stimulation may determine downstream signaling characteristics.
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Cálcio , Canais Iônicos , Cálcio/metabolismo , Sinalização do Cálcio , Canais Iônicos/genética , Canais Iônicos/metabolismo , Ligantes , Mecanotransdução Celular/fisiologiaRESUMO
Promoting the establishment of collateral circulation is essential for chronic lower extremity ischemia. However, no effective therapeutic drugs have yet been developed. Recent studies discovered that in the peripheral arteries, there are GABAB1 receptors expressed in endothelial cells and smooth muscle cells, these receptors may have some effects in regulating vascular functions, but the precise mechanism is not yet clear. This study explores the effect of GABAB1 receptor inhibition on angiogenesis and its regulatory mechanism. The expression of GABAB1 in HUVECs was knocked down using shRNA transfection, and effects in HUVECs' proliferation, migration, and tube formation ability were detected. Western blot and RT-PCR were used to verify the signal pathway. The murine hind limb ischemia model was used to verify the effect of CGP35348, an antagonist of GABAB1R, on the recovery of blood flow perfusion and angiogenesis in ischemic tissues. Cell proliferation, migration, and tube formation ability were improved after GABAB1 receptor knockdown in HUVECs. The phosphorylation of the HIPPO/YAP pathway decreased, while the effect of promoting angiogenesis increased. After treating the ischemic hindlimbs of mice with GABAB1 receptor antagonists, the blood flow perfusion recovered, and the angiogenesis increased. These findings demonstrate the effect of GABAB1 receptor inhibition on the HIPPO/YAP pathway in regulating angiogenesis, suggesting that inhibiting GABAB1 receptor levels might be a novel approach for chronic lower extremity ischemia diseases.
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PURPOSE: This study aimed to develop and internally validate nomograms for predicting restenosis after endovascular treatment of lower extremity arterial diseases. MATERIALS AND METHODS: A total of 181 hospitalized patients with lower extremity arterial disease diagnosed for the first time between 2018 and 2019 were retrospectively collected. Patients were randomly divided into a primary cohort (n=127) and a validation cohort (n=54) at a ratio of 7:3. The least absolute shrinkage and selection operator (LASSO) regression was used to optimize the feature selection of the prediction model. Combined with the best characteristics of LASSO regression, the prediction model was established by multivariate Cox regression analysis. The predictive models' identification, calibration, and clinical practicability were evaluated by the C index, calibration curve, and decision curve. The prognosis of patients with different grades was compared by survival analysis. Internal validation of the model used data from the validation cohort. RESULTS: The predictive factors included in the nomogram were lesion site, use of antiplatelet drugs, application of drug coating technology, calibration, coronary heart disease, and international normalized ratio (INR). The prediction model demonstrated good calibration ability, and the C index was 0.762 (95% confidence interval: 0.691-0.823). The C index of the validation cohort was 0.864 (95% confidence interval: 0.801-0.927), which also showed good calibration ability. The decision curve shows that when the threshold probability of the prediction model is more significant than 2.5%, the patients benefit significantly from our prediction model, and the maximum net benefit rate is 30.9%. Patients were graded according to the nomogram. Survival analysis found that there was a significant difference in the postoperative primary patency rate between patients of different classifications (log-rank p<0.001) in both the primary cohort and the validation cohort. CONCLUSION: We developed a nomogram to predict the risk of target vessel restenosis after endovascular treatment by considering information on lesion site, postoperative antiplatelet drugs, calcification, coronary heart disease, drug coating technology, and INR. CLINICAL IMPACT: Clinicians can grade patients after endovascular procedure according to the scores of the nomograms and apply intervention measures of different intensities for people at different risk levels. During the follow-up process, an individualized follow-up plan can be further formulated according to the risk classification. Identifying and analyzing risk factors is essential for making appropriate clinical decisions to prevent restenosis.
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OBJECTIVES: To identify the role and mechanism of a male specific gene, SRY, in I/R-induced hepatic injury. BACKGROUND: Males are more vulnerable to I/R injury than females. However, the mechanism of these sex-based differences remains poorly defined. METHODS: Clinicopathologic data of patients who underwent hepatic resection were identified from an international multi-institutional database. Liver specific SRY TG mice were generated, and subjected to I/R insult with their littermate WT controls in vivo. In vitro experiments were performed by treating primary hepatocytes from TG and WT mice with hypoxia/reoxygen-ation stimulation. RESULTS: Clinical data showed that postoperative aminotransferase level, incidence of overall morbidity and liver failure were markedly higher among 1267 male versus 508 female patients who underwent hepatic resection. SRY was dramatically upregulated during hepatic I/R injury. Overexpression of SRY in male TG mice and ectopic expression of SRY in female TG mice exacerbated liver I/R injury compared with WTs as manifested by increased inflammatory reaction, oxidative stress and cell death in vivo and in vitro. Mechanistically, SRY interacts with Glycogen synthase kinase-3ß (GSK-3ß) and ß-catenin, and promotes phosphorylation and degradation of ß-catenin, leading to suppression of the downstream FOXOs, and activation of NF-κBand TLR4 signaling. Furthermore, activation of ß-catenin almost completely reversed the SRYoverexpression-mediated exacerbation of hepatic I/R damage. CONCLUSIONS: SRY is a novel hepatic I/R mediator that promotes hepatic inflammatory reaction, oxidative stress and cell necrosis via inhibiting Wnt/ß-catenin signaling, which accounts for the sex-based disparity in hepatic I/R injuries.
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Hepatopatias , Traumatismo por Reperfusão , Proteína da Região Y Determinante do Sexo/metabolismo , Animais , Apoptose , Feminino , Glicogênio Sintase Quinase 3 beta/metabolismo , Isquemia , Fígado/patologia , Hepatopatias/metabolismo , Masculino , Camundongos , Caracteres Sexuais , beta CateninaRESUMO
In our previous studies, we found that T24 lung metastatic cancer cells showed high invasion and metastasis abilities and cancer stem cell characteristics compared with T24 primary cancer cells. By screening for the expression of CXC chemokines in both cell lines, we found that CXCL5 is highly expressed in T24-L cells. The aim of this study is to shed light on the relationship of CXCL5 with epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs). RNAi technology was used to decrease CXCL5 expression in the T24-L cell line, and the EMT and CSCs of the shCXCL5 group and the control group were compared. The CXCR2 inhibitor SB225002 was used to inhibit the receptor of CXCL5 to determine the effect of the CXCL5/CXCR2 axis. The knockdown of CXCL5 expression in T24-L cells reduced their EMT and CSC characteristics. RT-PCR and Western blot analyses revealed the downregulation of N-cadherin, Vimentin and CD44. In addition, when CD44 expression was knocked down, the EMT ability of the cells was also inhibited. This phenomenon was most pronounced when both CXCL5 and CD44 were knocked down. CXCL5 and CD44 can affect the EMT and stem cell capacity of T24-L cells through some interaction.
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Quimiocina CXCL5/genética , Receptores de Hialuronatos/genética , Neoplasias Pulmonares/patologia , Neoplasias da Bexiga Urinária/secundário , Caderinas/fisiologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Vimentina/fisiologiaRESUMO
INTRODUCTION: Chronic limb threat ischemia is associated with cardiovascular events, resulting in high amputation, morbidity and mortality rates. This study aims to accomplish a comprehensive summary of randomized controlled trials and single-center trials related to drug-coated balloons (DCBs) in the treatment of below-the-knee (BTK) artery disease, and to provide a recommendation for the application of DCBs in BTK artery disease. METHODS: Five electronic databases were used to retrieve relevant articles on the safety and effectiveness of DCBs in the treatment of BTK artery disease. A random-effects model was applied to calculate the standard mean deviation, odds ratio (OR) and their 95% of confidence interval (CI). RESULTS: As of April 8, 2021, a total of 241 articles were retrieved, but only 13 articles were finally included for meta-analysis. The 12 mo follow-up study found that major adverse events , all-cause mortality, major amputation ,and target lesion revascularization had no statistically significant difference between the DCBs group and the control group (target lesion revascularization: OR = 0.68, 95% CI: 0.36, 1.31; all-cause mortality: OR = 1.30, 95% CI: 0.69, 2.46; major amputation: OR = 1.34, 95% CI: 0.64, 2.79; target lesion revascularization: OR = 0.72, 95% CI: 0.35, 1.45). CONCLUSIONS: The meta-analysis results of randomized controlled trials focusing on comparing DCBs and other treatments suggest that DCBs do not have significant advantages in the treatment of BTK artery disease when compare with percutaneous transluminal angioplasty (PTA), but better than control intervention except PTA in both safety and efficacy end points. However, the results of meta-analysis of single-arm trial reported DCBs in treating BTK artery lesions are significantly improved compared with the meta-analysis concentrating on PTA.
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Angioplastia com Balão , Doença Arterial Periférica , Angioplastia com Balão/efeitos adversos , Angioplastia com Balão/métodos , Materiais Revestidos Biocompatíveis , Artéria Femoral/cirurgia , Seguimentos , Humanos , Isquemia/terapia , Paclitaxel/efeitos adversos , Doença Arterial Periférica/cirurgia , Artéria Poplítea/cirurgia , Ensaios Clínicos Controlados Aleatórios como Assunto , Fatores de Tempo , Resultado do Tratamento , Grau de Desobstrução VascularRESUMO
BACKGROUND: Patients with critical limb ischemia (CLI) are at great risk of major amputation and cardiovascular events. Adipose-derived mesenchymal stem cell (ADSC) therapy is a promising therapeutic strategy for CLI, but the poor engraftment and insufficient angiogenic ability of ADSCs limit their regenerative potential. Herein, we explored the potential of human umbilical vein endothelial cells (HUVECs)-derived small extracellular vesicles (sEVs) for enhancing the therapeutic efficacy of ADSCs in CLI. RESULTS: sEVs derived from hypoxic HUVECs enhanced the resistance of ADSCs to reactive oxygen species (ROS) and further improved the proangiogenic ability of ADSCs in vitro. We found that the hypoxic environment altered the composition of sEVs from HUVECs and that hypoxia increased the level of miR-486-5p in sEVs. Compared to normoxic sEVs (nsEVs), hypoxic sEVs (hsEVs) of HUVECs significantly downregulated the phosphatase and tensin homolog (PTEN) via direct targeting of miR-486-5p, therefore activating the AKT/MTOR/HIF-1α pathway and influencing the survival and pro-angiogenesis ability of ADSCs. In a hindlimb ischemia model, we discovered that hsEVs-primed ADSCs exhibited superior cell engraftment, and resulted in better angiogenesis and tissue repair. CONCLUSION: hsEVs could be used as a therapeutic booster to improve the curative potential of ADSCs in a limb ischemia model. This finding offers new insight for CLI treatment.
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Vesículas Extracelulares , Células-Tronco Mesenquimais , MicroRNAs , Tecido Adiposo/metabolismo , Animais , Vesículas Extracelulares/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Hipóxia/metabolismo , Isquemia/metabolismo , Isquemia/terapia , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Neovascularização Patológica/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Tensinas/metabolismoRESUMO
OBJECTIVE: To determine the risk for pulmonary embolism (PE) and explore the relationship between the site of thrombosis and PE in patients with acute lower extremity deep vein thrombosis (DVT). METHODS: A total of 1585 hospitalized patients first diagnosed with acute lower extremity DVT were investigated retrospectively. The patients were divided into two groups: the non-PE group (Group 1) and the PE group (Group 2). Then, Group 2 was divided into two subgroups: asymptomatic pulmonary embolism (asPE, Group 2a) and symptomatic pulmonary embolism (sPE, Group 2b). Kaplan-Meier curves and logistic regression analysis were used to explore the relevant risk factors for PE. RESULTS: Among 1585 patients, 458 patients suffered from PE, accounting for 28.9%. 102 (22.3%) of them had the typical clinical manifestations of PE and were defined as sPE, and the remaining 356 (77.7%) patients were classified as asPE. Patients with proximal lower extremity DVT were significantly more predominant in the PE group than in the non-PE group (92.8% vs. 86.2%, P<0.001). Moreover, in Group 2, patients with typical PE manifestations showed a higher proportion of patients with right lower extremity DVT than left lower extremity DVT (26.7% vs. 17.7%, P = 0.035), and bilateral lower extremity DVT than unilateral DVT (44.1% vs. 20.5%, P<0.001). By multivariate analysis, alcohol consumption (OR, 1.824; 95% CI, 1.194-2.787; P = 0.005), heart failure (OR, 2.345; 95% CI, 1.560-3.526; P<0.001), proximal DVT (OR, 2.096; 95% CI,1.407-3.123; P<0.001) were independent risk factors for PE. CONCLUSIONS: Patients with proximal acute lower extremity DVT were more likely to suffer from PE than those with distal DVT. Patients with right acute lower extremity DVT had a higher risk of sPE than patients with left acute lower extremity DVT. Alcohol consumption and heart failure were associated with the occurrence of PE in patients with acute lower extremity DVT.
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Embolia Pulmonar , Trombose Venosa , Humanos , Extremidade Inferior/irrigação sanguínea , Embolia Pulmonar/complicações , Embolia Pulmonar/diagnóstico , Embolia Pulmonar/epidemiologia , Estudos Retrospectivos , Fatores de Risco , Resultado do Tratamento , Trombose Venosa/complicações , Trombose Venosa/epidemiologiaRESUMO
The dramatic reorganization of chromatin during mitosis is perhaps one of the most fundamental of all cell processes. It remains unclear how epigenetic histone modifications, despite their crucial roles in regulating chromatin architectures, are dynamically coordinated with chromatin reorganization in controlling this process. We have developed and characterized biosensors with high sensitivity and specificity based on fluorescence resonance energy transfer (FRET). These biosensors were incorporated into nucleosomes to visualize histone H3 Lys-9 trimethylation (H3K9me3) and histone H3 Ser-10 phosphorylation (H3S10p) simultaneously in the same live cell. We observed an anticorrelated coupling in time between H3K9me3 and H3S10p in a single live cell during mitosis. A transient increase of H3S10p during mitosis is accompanied by a decrease of H3K9me3 that recovers before the restoration of H3S10p upon mitotic exit. We further showed that H3S10p is causatively critical for the decrease of H3K9me3 and the consequent reduction of heterochromatin structure, leading to the subsequent global chromatin reorganization and nuclear envelope dissolution as a cell enters mitosis. These results suggest a tight coupling of H3S10p and H3K9me3 dynamics in the regulation of heterochromatin dissolution before a global chromatin reorganization during mitosis.
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Técnicas Biossensoriais/métodos , Montagem e Desmontagem da Cromatina , Código das Histonas , Proteínas de Bactérias , Montagem e Desmontagem da Cromatina/genética , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde , Células HEK293 , Heterocromatina/genética , Heterocromatina/metabolismo , Código das Histonas/genética , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas Luminescentes , Mitose , Modelos Biológicos , Análise de Célula Única/métodosRESUMO
Iatrogenic femoral artery pseudoaneurysm caused by invasive procedures is one of the common complications for endovascular interventions. We present a case of a young male with a complex iatrogenic femoral artery pseudoaneurysm as a result of iatrogenic femoral artery puncture. The defective femoral artery was repaired with combined bovine pericardial tube and autologous great saphenous vein grafts. Computed tomography angiography showed the grafts were still patent one year after the surgery.
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Falso Aneurisma/etiologia , Falso Aneurisma/cirurgia , Procedimentos Endovasculares/efeitos adversos , Artéria Femoral/lesões , Artéria Femoral/cirurgia , Doença Iatrogênica , Complicações Intraoperatórias/etiologia , Complicações Intraoperatórias/cirurgia , Pericárdio/transplante , Veia Safena/transplante , Procedimentos Cirúrgicos Vasculares/métodos , Ferimentos Penetrantes/etiologia , Ferimentos Penetrantes/cirurgia , Adulto , Falso Aneurisma/diagnóstico por imagem , Animais , Bovinos , Angiografia por Tomografia Computadorizada , Procedimentos Endovasculares/métodos , Artéria Femoral/diagnóstico por imagem , Humanos , Masculino , Transplante Autólogo , Transplante Heterólogo , Resultado do TratamentoRESUMO
Iatrogenic femoral artery pseudoaneurysm is a common complication of the endovascular procedures. Manual compression and thrombin injection are the conventional techniques to occlude the pseudoaneurysms. However, there are still some failed cases that applied these treatment options. The aim of the study is to seek a potential and alternative method with ProGlide system to close the pseudoaneurysm. During April 2018 to February 2019, 2 patients with iatrogenic pseudoaneurysm of the superficial femoral were treated with the suture-base closure device--ProGlide. After punctured the pseudoaneurysm and placed a 6-F sheath, the guide wire was placed in the right femoral artery via the access of the pseudoaneurysm neck. Then the pseudoaneurysm neck was sutured by ProGlide to occlude the blood supply to the pseudoaneurysm. These 2 patients were cured with no complications and complaints, which revealed that percutaneous suture technique with ProGlide at the neck level of pseudoaneurysm provides a novel method for the management of vascular access pseudoaneurysm, especially in those with a wide and short neck.
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Falso Aneurisma/cirurgia , Artéria Femoral/cirurgia , Complicações Pós-Operatórias/cirurgia , Técnicas de Sutura , Adulto , Procedimentos Endovasculares , Humanos , Doença Iatrogênica , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
Beneficial effects of metformin on cancer risk and mortality have been proved by epidemiological and clinical studies, thus attracting research interest in elucidating the underlying mechanisms. Recently, tumour-associated macrophages (TAMs) appeared to be implicated in metformin-induced antitumour activities. However, how metformin inhibits TAMs-induced tumour progression remains ill-defined. Here, we report that metformin-induced antitumour and anti-angiogenic activities were not or only partially contributed by its direct inhibition of functions of tumour and endothelial cells. By skewing TAM polarization from M2- to M1-like phenotype, metformin inhibited both tumour growth and angiogenesis. Depletion of TAMs by clodronate liposomes eliminated M2-TAMs-induced angiogenic promotion, while also abrogating M1-TAMs-mediated anti-angiogenesis, thus promoting angiogenesis in tumours from metformin treatment mice. Further in vitro experiments using TAMs-conditioned medium and a coculture system were performed, which demonstrated an inhibitory effect of metformin on endothelial sprouting and tumour cell proliferation promoted by M2-polarized RAW264.7 macrophages. Based on these results, metformin-induced inhibition of tumour growth and angiogenesis is greatly contributed by skewing of TAMs polarization in microenvironment, thus offering therapeutic opportunities for metformin in cancer treatment.
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Substantial data from preclinical studies have revealed the biphasic effects of statins on cardiovascular angiogenesis. Although some have reported the anti-angiogenic potential of statins in malignant tumors, the underlying mechanism remains poorly understood. The aim of this study is to elucidate the mechanism by which simvastatin, a member of the statin family, inhibits tumor angiogenesis. Simvastatin significantly suppressed tumor cell-conditioned medium-induced angiogenic promotion in vitro, and resulted in dose-dependent anti-angiogenesis in vivo. Further genetic silencing of hypoxia-inducible factor-1α (HIF-1α) reduced vascular endothelial growth factor and fibroblast growth factor-2 expressions in 4T1 cells and correspondingly ameliorated HUVEC proliferation facilitated by tumor cell-conditioned medium. Additionally, simvastatin induced angiogenic inhibition through a mechanism of post-transcriptional downregulation of HIF-1α by increasing the phosphorylation level of AMP kinase. These results were further validated by the fact that 5-aminoimidazole-4-carboxamide ribonucleotide reduced HIF-1α protein levels and ameliorated the angiogenic ability of endothelial cells in vitro and in vivo. Critically, inhibition of AMPK phosphorylation by compound C almost completely abrogated simvastatin-induced anti-angiogenesis, which was accompanied by the reduction of protein levels of HIF-1α and its downstream pro-angiogenic factors. These findings reveal the mechanism by which simvastatin induces tumor anti-angiogenesis, and therefore identifies the target that explains the beneficial effects of statins on malignant tumors.
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Proteínas Quinases Ativadas por AMP/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Neovascularização Patológica/prevenção & controle , Sinvastatina/farmacologia , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Feminino , Fator 2 de Crescimento de Fibroblastos/análise , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos BALB C , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
PURPOSE: To compare outcomes of patients who received simultaneous tributary endovenous laser ablation (EVLA) or foam sclerotherapy (FS) with EVLA of the great saphenous vein (GSV) trunk. METHODS AND MATERIALS: This study recruited 418 patients (542 legs) with diagnosed varicose veins. Patients in the EVLA/FS group (255 patients, 327 legs) received concomitant FS for the tributaries with truncal lasering. For the EVLA-alone group (163 patients, 215 legs), tributaries (8W) were ablated with EVLA in addition to the GSV trunk (14W). Complications, Aberdeen Varicose Vein Questionnaire (AVVQ), EuroQol Group 5-Dimension Self-Report Questionnaire (EQ-5D), numerical rating scale (NRS) scores, and condition of residual varicosities were assessed at 3 days, 4 weeks, and 6 months after procedure. All residual varicosities were identified and treated with a staged FS at 6 months. RESULTS: Except for ecchymosis, incidence of other complications was not significantly different between both groups at 6 months. Pain NRS scores of the EVLA/FS group were remarkably elevated at 4 weeks and then, at 6 months, declined to a level similar to the EVLA-alone group. The EVLA/FS group exhibited more significant improvement in both AVVQ and EQ-5D scales than the EVLA group at 6 months, while exhibiting poor improvement at 4 weeks. The EVLA/FS group had a significantly lower rate of residual varicosities than the EVLA group, thus reducing the need for the staged FS. CONCLUSIONS: These results confirm the feasibility and safety of simultaneous tributary EVLA and FS. In addition, they indicate better early quality-of-life improvement and a reduced reoperation rate of simultaneously combined truncal EVLA and tributary FS.
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Terapia a Laser/métodos , Perna (Membro)/irrigação sanguínea , Veia Safena , Escleroterapia/métodos , Varizes/terapia , Terapia Combinada , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Medição da Dor , Estudos Prospectivos , Inquéritos e Questionários , Resultado do Tratamento , Ultrassonografia de IntervençãoRESUMO
BACKGROUND AND STUDY AIMS: To demonstrate the clinical use of a multimodal endoscope with a targeted fluorescently labeled peptide for quantitative detection of Barrett's neoplasia. PATIENTS AND METHODS: We studied 50 patients with Barrett's esophagus using a prototype multimodal endoscope with a fluorescently labeled peptide. Co-registered fluorescence and reflectance images were converted to ratios to correct for differences in distance and geometry over the image field of view. The ratio images were segmented using a unique threshold that maximized the variance between high and low intensities to localize regions of high grade dysplasia (HGD) and esophageal adenocarcinoma (EAC). RESULTS: Early neoplasia (HGD and EAC) was identified with 94â% specificity and 96â% positive predictive value at a threshold of 1.49.âThe mean results for HGD and EAC were significantly greater than those for squamous/Barrett's esophagus and low grade dysplasia by one-way analysis of variance (ANOVA). The receiver operator characteristic curve for detection of early neoplasia had an area under the curve of 0.884.âNo adverse events associated with the endoscope or peptide were found. CONCLUSION: A multimodal endoscope can quantify fluorescence images from targeted peptides to localize early Barrett's neoplasia. (ClinicalTrials.gov number NCT01630798.).
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Adenocarcinoma/diagnóstico , Esôfago de Barrett/diagnóstico , Detecção Precoce de Câncer/métodos , Endoscópios Gastrointestinais , Neoplasias Esofágicas/diagnóstico , Esôfago/patologia , Imagem Multimodal/instrumentação , Lesões Pré-Cancerosas , Adulto , Idoso , Idoso de 80 Anos ou mais , Diagnóstico Diferencial , Desenho de Equipamento , Feminino , Fluorescência , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos TestesRESUMO
Matrix mechanics controls cell fate by modulating the bonds between integrins and extracellular matrix (ECM) proteins. However, it remains unclear how fibronectin (FN), type 1 collagen, and their receptor integrin subtypes distinctly control force transmission to regulate focal adhesion kinase (FAK) activity, a crucial molecular signal governing cell adhesion/migration. Here we showed, using a genetically encoded FAK biosensor based on fluorescence resonance energy transfer, that FN-mediated FAK activation is dependent on the mechanical tension, which may expose its otherwise hidden FN synergy site to integrin α5. In sharp contrast, the ligation between the constitutively exposed binding motif of type 1 collagen and its receptor integrin α2 was surprisingly tension-independent to induce sufficient FAK activation. Although integrin α subunit determines mechanosensitivity, the ligation between α subunit and the ECM proteins converges at the integrin ß1 activation to induce FAK activation. We further discovered that the interaction of the N-terminal protein 4.1/ezrin/redixin/moesin basic patch with phosphatidylinositol 4,5-biphosphate is crucial during cell adhesion to maintain the FAK activation from the inhibitory effect of nearby protein 4.1/ezrin/redixin/moesin acidic sites. Therefore, different ECM proteins either can transmit or can shield from mechanical forces to regulate cellular functions, with the accessibility of ECM binding motifs by their specific integrin α subunits determining the biophysical mechanisms of FAK activation during mechanotransduction.
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Proteínas do Citoesqueleto/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Mecanotransdução Celular/fisiologia , Sítios de Ligação/genética , Fenômenos Biomecânicos , Linhagem Celular Tumoral , Colágeno Tipo I/metabolismo , Fibronectinas/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Processamento de Imagem Assistida por Computador , Immunoblotting , Imunoprecipitação , Integrina alfa2/genética , Integrina alfa2/metabolismoRESUMO
We developed a quantum-dot-based fluorescence resonance energy transfer (QD-FRET) nanosensor to visualize the activity of matrix metalloproteinase (MT1-MMP) at cell membrane. A bended peptide with multiple motifs was engineered to position the FRET pair at a close proximity to allow energy transfer, which can be cleaved by active MT1-MMP to result in FRET changes and the exposure of cell penetrating sequence. Via FRET and penetrated QD signals, the nanosensor can profile cancer cells.
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Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Neoplasias/enzimologia , Peptídeos/metabolismo , Análise de Célula Única/métodos , Sequência de Aminoácidos , Técnicas Biossensoriais/métodos , Linhagem Celular Tumoral , Feminino , Corantes Fluorescentes/química , Células HeLa , Humanos , Metaloproteinase 14 da Matriz/análise , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Pontos Quânticos/química , Pontos Quânticos/metabolismoRESUMO
OBJECTIVE: To develop a peptide probe that could be used for gastric cancer detection via binding to CD44 protein with specificity and affinity. RESULTS: A 12-mer phage peptide library was screened against immobilized CD44 protein. Bound phage counts using ELISA were performed to identify phage clones carrying the most highly selective peptide, which termed RP-1. Immunofluorescence and flow cytometry analysis indicated that the consensus peptide RP-1 could bind to CD44-positive gastric cancer cells with mean fluorescence intensities significantly higher than that of CD44-negative cells. CD44 knockdown led to decreased binding activity of RP-1 to the same cell line. Tissue array technique was used to identify the relationship (r = 0.556) between peptide binding and CD44 detection on gastric cancer tissues. Further, the hyaluronan-binding domain of CD44 was docked with RP-1 using computer modeling/docking approaches, revealing a RP-1/CD44 interaction with geometrical and energy match (-8.6 kcal/mol). CONCLUSIONS: The RP-1 peptide we screened exhibits affinity and specificity to CD44 on cells and has the potential to be used as a candidate probe for gastric cancer cell targeting.
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Receptores de Hialuronatos/química , Receptores de Hialuronatos/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Neoplasias Gástricas/química , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Simulação de Acoplamento Molecular , Biblioteca de Peptídeos , Peptídeos/genética , Neoplasias Gástricas/metabolismoRESUMO
Cell invasion and migration that occurs, for example, in cancer metastasis is rooted in the ability of cells to navigate through varying levels of physical constraint exerted by the extracellular matrix. Cancer cells can invade matrices in either a protease-independent or a protease-dependent manner. An emerging critical component that influences the mode of cell invasion is the traction stresses generated by the cells in response to the physicostructural properties of the extracellular matrix. In this study, we have developed a reference-free quantitative assay for measuring three-dimensional (3D) traction stresses generated by cells during the initial stages of invasion into matrices exerting varying levels of mechanical resistance. Our results show that as cells encounter higher mechanical resistance, a larger fraction of them shift to protease-mediated invasion, and this process begins at lower values of cell invasion depth. On the other hand, the compressive stress generated by the cells at the onset of protease-mediated invasion is found to be independent of matrix stiffness, suggesting that 3D traction stress is a key factor in triggering protease-mediated cancer cell invasion. At low 3D compressive traction stresses, cells utilize bleb formation to indent the matrix in a protease independent manner. However, at higher stress values, cells utilize invadopodia-like structures to mediate protease-dependent invasion into the 3D matrix. The critical value of compressive traction stress at the transition from a protease-independent to a protease-dependent mode of invasion is found to be â¼165 Pa.
Assuntos
Neoplasias/patologia , Peptídeo Hidrolases/metabolismo , Estresse Fisiológico , Fenômenos Biomecânicos/efeitos dos fármacos , Linhagem Celular Tumoral , Colágeno/farmacologia , Combinação de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Humanos , Laminina/farmacologia , Invasividade Neoplásica , Proteoglicanas/farmacologia , Análise de Célula Única , Estresse Fisiológico/efeitos dos fármacosRESUMO
This study investigated the impact of cadherin binding differences on both cell sorting and GTPase activation. The use of N-terminal domain point mutants of Xenopus C-cadherin enabled us to quantify binding differences and determine their effects on cadherin-dependent functions without any potential complications arising as a result of differences in cytodomain interactions. Dynamic cell-cell binding measurements carried out with the micropipette manipulation technique quantified the impact of these mutations on the two-dimensional binding affinities and dissociation rates of cadherins in the native context of the cell membrane. Pairwise binding affinities were compared with in vitro cell-sorting specificity and ligation-dependent GTPase signaling. Two-dimensional affinity differences greater than five-fold correlated with cadherin-dependent in vitro cell segregation, but smaller differences failed to induce cell sorting. Comparison of the binding affinities with GTPase signaling amplitudes further demonstrated that differential binding also proportionally modulates intracellular signaling. These results show that differential cadherin affinities have broader functional consequences than merely controlling cell-cell cohesion.