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BACKGROUND: Important clinical information of patients is present in unstructured free-text fields of Electronic Health Records (EHRs). While this information can be extracted using clinical Natural Language Processing (cNLP), the recognition of negation modifiers represents an important challenge. A wide range of cNLP applications have been developed to detect the negation of medical entities in clinical free-text, however, effective solutions for languages other than English are scarce. This study aimed at developing a solution for negation recognition in Spanish EHRs based on a combination of a customized rule-based NegEx layer and a convolutional neural network (CNN). METHODS: Based on our previous experience in real world evidence (RWE) studies using information embedded in EHRs, negation recognition was simplified into a binary problem ('affirmative' vs. 'non-affirmative' class). For the NegEx layer, negation rules were obtained from a publicly available Spanish corpus and enriched with custom ones, whereby the CNN binary classifier was trained on EHRs annotated for clinical named entities (cNEs) and negation markers by medical doctors. RESULTS: The proposed negation recognition pipeline obtained precision, recall, and F1-score of 0.93, 0.94, and 0.94 for the 'affirmative' class, and 0.86, 0.84, and 0.85 for the 'non-affirmative' class, respectively. To validate the generalization capabilities of our methodology, we applied the negation recognition pipeline on EHRs (6,710 cNEs) from a different data source distribution than the training corpus and obtained consistent performance metrics for the 'affirmative' and 'non-affirmative' class (0.95, 0.97, and 0.96; and 0.90, 0.83, and 0.86 for precision, recall, and F1-score, respectively). Lastly, we evaluated the pipeline against two publicly available Spanish negation corpora, the IULA and NUBes, obtaining state-of-the-art metrics (1.00, 0.99, and 0.99; and 1.00, 0.93, and 0.96 for precision, recall, and F1-score, respectively). CONCLUSION: Negation recognition is a source of low precision in the retrieval of cNEs from EHRs' free-text. Combining a customized rule-based NegEx layer with a CNN binary classifier outperformed many other current approaches. RWE studies highly benefit from the correct recognition of negation as it reduces false positive detections of cNE which otherwise would undoubtedly reduce the credibility of cNLP systems.
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Algoritmos , Processamento de Linguagem Natural , Humanos , Redes Neurais de Computação , Registros Eletrônicos de Saúde , IdiomaRESUMO
OBJECTIVE: Experimental studies link oscillatory flow accompanied by flow reversal to impaired endothelial cell function. The relation of flow reversal with vascular function and arterial stiffness remains incompletely defined. APPROACH AND RESULTS: We measured brachial diastolic flow patterns along with vasodilator function in addition to tonometry-based central and peripheral arterial stiffness in 5708 participants (age 47±13 years, 53% women) in the Framingham Heart Study Offspring and Third Generation cohorts. Brachial artery diastolic flow reversal was present in 35% of the participants. In multivariable regression models, the presence of flow reversal was associated with lower flow-mediated dilation (3.9±0.2 versus 5.0±0.2%; P<0.0001) and reactive hyperemic flow velocity (50±0.99 versus 57±0.93 cm/s; P<0.0001). The presence of flow reversal (compared with absence) was associated with higher central aortic stiffness (carotid-femoral pulse wave velocity 9.3±0.1 versus 8.9±0.1 m/s), lower muscular artery stiffness (carotid-radial pulse wave velocity 9.6±0.1 versus 9.8±0.1 m/s), and higher forearm vascular resistance (5.32±0.03 versus 4.66±0.02 log dyne/s/cm5; P<0.0001). The relations of diastolic flow velocity with flow-mediated dilation, aortic stiffness, and forearm vascular resistance were nonlinear, with a steeper decline in vascular function associated with increasing magnitude of flow reversal. CONCLUSIONS: In our large, community-based sample, brachial artery flow reversal was common and associated with impaired vasodilator function and higher aortic stiffness. Our findings are consistent with the concept that flow reversal may contribute to vascular dysfunction.
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Artéria Braquial/fisiopatologia , Doenças Cardiovasculares/fisiopatologia , Endotélio Vascular/fisiopatologia , Antebraço/irrigação sanguínea , Fluxo Pulsátil , Rigidez Vascular , Vasodilatação , Adulto , Idoso , Aorta/fisiopatologia , Velocidade do Fluxo Sanguíneo , Artéria Braquial/diagnóstico por imagem , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/etiologia , Estudos Transversais , Endotélio Vascular/diagnóstico por imagem , Feminino , Humanos , Hiperemia/fisiopatologia , Modelos Lineares , Masculino , Manometria , Massachusetts , Pessoa de Meia-Idade , Análise Multivariada , Dinâmica não Linear , Razão de Chances , Fluxo Sanguíneo Regional , Fatores de Risco , Ultrassonografia Doppler de Pulso , Resistência VascularRESUMO
OBJECTIVE: Endothelial dysfunction is linked to insulin resistance, inflammatory activation, and increased cardiovascular risk in diabetes mellitus; however, the mechanisms remain incompletely understood. Recent studies have identified proinflammatory signaling of wingless-type family member (Wnt) 5a through c-jun N-terminal kinase (JNK) as a regulator of metabolic dysfunction with potential relevance to vascular function. We sought to gain evidence that increased activation of Wnt5a-JNK signaling contributes to impaired endothelial function in patients with diabetes mellitus. APPROACH AND RESULTS: We measured flow-mediated dilation of the brachial artery and characterized freshly isolated endothelial cells by protein expression, eNOS activation, and nitric oxide production in 85 subjects with type 2 diabetes mellitus (n=42) and age- and sex-matched nondiabetic controls (n=43) and in human aortic endothelial cells treated with Wnt5a. Endothelial cells from patients with diabetes mellitus displayed 1.3-fold higher Wnt5a levels (P=0.01) along with 1.4-fold higher JNK activation (P<0.01) without a difference in total JNK levels. Higher JNK activation was associated with lower flow-mediated dilation, consistent with endothelial dysfunction (r=0.53, P=0.02). Inhibition of Wnt5a and JNK signaling restored insulin and A23187-mediated eNOS activation and improved nitric oxide production in endothelial cells from patients with diabetes mellitus. In endothelial cells from nondiabetic controls, rWnt5a treatment inhibited eNOS activation replicating the diabetic endothelial phenotype. In human aortic endothelial cells, Wnt5a-induced impairment of eNOS activation and nitric oxide production was reversed by Wnt5a and JNK inhibition. CONCLUSIONS: Our findings demonstrate that noncanonical Wnt5a signaling and JNK activity contribute to vascular insulin resistance and endothelial dysfunction and may represent a novel therapeutic opportunity to protect the vasculature in patients with diabetes mellitus.
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Artéria Braquial/enzimologia , Diabetes Mellitus Tipo 2/enzimologia , Células Endoteliais/enzimologia , Endotélio Vascular/enzimologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Vasodilatação , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Adulto , Idoso , Artéria Braquial/efeitos dos fármacos , Artéria Braquial/fisiopatologia , Estudos de Casos e Controles , Células Cultivadas , Diabetes Mellitus Tipo 2/fisiopatologia , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Ativação Enzimática , Feminino , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/farmacologia , Vasodilatação/efeitos dos fármacos , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt-5aRESUMO
OBJECTIVE: Prior studies demonstrate mitochondrial dysfunction with increased reactive oxygen species generation in peripheral blood mononuclear cells in diabetes mellitus. Oxidative stress-mediated damage to mitochondrial DNA promotes atherosclerosis in animal models. Thus, we evaluated the relation of mitochondrial DNA damage in peripheral blood mononuclear cells s with vascular function in patients with diabetes mellitus and with atherosclerotic cardiovascular disease. APPROACH AND RESULTS: We assessed non-invasive vascular function and mitochondrial DNA damage in 275 patients (age 57 ± 9 years, 60 % women) with atherosclerotic cardiovascular disease alone (N = 55), diabetes mellitus alone (N = 74), combined atherosclerotic cardiovascular disease and diabetes mellitus (N = 48), and controls age >45 without diabetes mellitus or atherosclerotic cardiovascular disease (N = 98). Mitochondrial DNA damage measured by quantitative PCR in peripheral blood mononuclear cells was higher with clinical atherosclerosis alone (0.55 ± 0.65), diabetes mellitus alone (0.65 ± 1.0), and combined clinical atherosclerosis and diabetes mellitus (0.89 ± 1.32) as compared to control subjects (0.23 ± 0.64, P < 0.0001). In multivariable models adjusting for age, sex, and relevant cardiovascular risk factors, clinical atherosclerosis and diabetes mellitus remained associated with higher mitochondrial DNA damage levels (ß = 0.14 ± 0.13, P = 0.04 and ß = 0.21 ± 0.13, P = 0.002, respectively). Higher mitochondrial DNA damage was associated with higher baseline pulse amplitude, a measure of arterial pulsatility, but not with flow-mediated dilation or hyperemic response, measures of vasodilator function. CONCLUSIONS: We found greater mitochondrial DNA damage in patients with diabetes mellitus and clinical atherosclerosis. The association of mitochondrial DNA damage and baseline pulse amplitude may suggest a link between mitochondrial dysfunction and excessive small artery pulsatility with potentially adverse microvascular impact.
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Aterosclerose/genética , DNA Mitocondrial/genética , Diabetes Mellitus Tipo 2/genética , Leucócitos Mononucleares/metabolismo , Adulto , Idoso , Aterosclerose/complicações , Aterosclerose/metabolismo , Velocidade do Fluxo Sanguíneo/genética , Artéria Braquial/fisiopatologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Feminino , Humanos , Hiperemia/genética , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/genética , Fatores de RiscoRESUMO
Obesity is associated with the development of vascular insulin resistance; however, pathophysiological mechanisms are poorly understood. We sought to investigate the role of WNT5A-JNK in the regulation of insulin-mediated vasodilator responses in human adipose tissue arterioles prone to endothelial dysfunction. In 43 severely obese (BMI 44±11 kg/m2) and five metabolically normal non-obese (BMI 26±2 kg/m2) subjects, we isolated arterioles from subcutaneous and visceral fat during planned surgeries. Using videomicroscopy, we examined insulin-mediated, endothelium-dependent vasodilator responses and characterized adipose tissue gene and protein expression using real-time polymerase chain reaction and Western blot analyses. Immunofluorescence was used to quantify endothelial nitric oxide synthase (eNOS) phosphorylation. Insulin-mediated vasodilation was markedly impaired in visceral compared to subcutaneous vessels from obese subjects (p<0.001), but preserved in non-obese individuals. Visceral adiposity was associated with increased JNK activation and elevated expression of WNT5A and its non-canonical receptors, which correlated negatively with insulin signaling. Pharmacological JNK antagonism with SP600125 markedly improved insulin-mediated vasodilation by sixfold (p<0.001), while endothelial cells exposed to recombinant WNT5A developed insulin resistance and impaired eNOS phosphorylation (p<0.05). We observed profound vascular insulin resistance in the visceral adipose tissue arterioles of obese subjects that was associated with up-regulated WNT5A-JNK signaling and impaired endothelial eNOS activation. Pharmacological JNK antagonism markedly improved vascular endothelial function, and may represent a potential therapeutic target in obesity-related vascular disease.
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Adiposidade , Arteríolas/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Resistência à Insulina , Insulina/farmacologia , Gordura Intra-Abdominal/irrigação sanguínea , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Obesidade/enzimologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt-5a/metabolismo , Adolescente , Adulto , Arteríolas/enzimologia , Arteríolas/fisiopatologia , Estudos de Casos e Controles , Células Cultivadas , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiopatologia , Feminino , Humanos , Técnicas In Vitro , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo III/metabolismo , Obesidade/fisiopatologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Adulto JovemRESUMO
Endothelial cells in the vascular system are constantly subjected to the frictional force of shear stress due to the pulsatile nature of blood flow. Although several proteins form part of the shear stress mechano-sensing pathway, the identification of mechano-transducing pathways is largely unknown. Given the increasing evidence for a signaling function of mitochondria in endothelial cells, the aim of this study was to investigate their role as mechano-sensor organelles during laminar shear stress (LSS). We demonstrated that LSS activates intracellular signaling pathways that modulate not only mitochondrial dynamics but also mitochondrial function. At early time points of LSS, the fission-related protein Drp1 was recruited from the cytosol to mitochondria and activated mitochondrial fission. LSS-dependent increase in intracellular Ca(2+) concentration was indispensable for mitochondrial fission. As alterations in mitochondrial dynamics have been related to changes in bioenergetics profiles, we studied mitochondrial function after LSS. We found that LSS decreased respiration rate, increased mitochondrial membrane potential and promoted the mitochondrial generation of ROS with the subsequent oxidation and activation of the antioxidant enzyme PRX3. Our data support a novel and active role for mitochondria in endothelial cells as active players, able to transduce the mechanical force of shear stress in the vascular endothelium into a biological response.
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ADP plays critical signaling roles in the vascular endothelium. ADP receptors are targeted by several cardiovascular drugs, yet the intracellular pathways modulated by ADP are incompletely understood. These studies have identified important roles for the phosphatase PTEN in ADP-dependent modulation of the endothelial isoform of nitric oxide synthase (eNOS) as well as of lipid and protein kinase pathways in endothelial cells. We find that ADP-promoted eNOS activation as well as phosphorylation of p38 MAPK are enhanced by siRNA-mediated PTEN knockdown. However, the increase in ADP-dependent eNOS activation promoted by PTEN knockdown is abrogated by siRNA-mediated knockdown of p38 MAPK. These findings indicate that PTEN tonically suppresses both p38 phosphorylation as well as ADP-stimulated eNOS activity. A key enzymatic activity of PTEN is its role as a lipid phosphatase, catalyzing the dephosphorylation of phosphoinositol-3,4,5-trisphosphate (PIP3) to phosphoinositol-4,5-bisphosphate (PIP2). We performed biochemical analyses of cellular phospholipids in endothelial cells to show that siRNA-mediated PTEN knockdown leads to a marked increase in PIP3. Because these complex lipids activate the small GTPase Rac1, we explored the role of PTEN in ADP-modulated Rac1 activation. We used a FRET biosensor for Rac1 to show that ADP-dependent Rac1 activation is blocked by siRNA-mediated PTEN knockdown. We then exploited a FRET biosensor for PIP3 to show that the striking ADP-dependent increase in intracellular PIP3 is entirely blocked by PTEN knockdown. These studies identify a key role for PTEN in the modulation of lipid mediators involved in ADP receptor-regulated endothelial signaling pathways involving eNOS activation in vascular endothelial cells.
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Difosfato de Adenosina/farmacologia , Células Endoteliais/enzimologia , PTEN Fosfo-Hidrolase/metabolismo , Transdução de Sinais/efeitos dos fármacos , Actinas/metabolismo , Animais , Aorta/citologia , Bovinos , Movimento Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Modelos Biológicos , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Efforts in the pharmaceutical market have been aimed at ensuring that the benefits obtained from the introduction of new therapies justify the associated costs. In recent years, drug payment models in healthcare have undergone a dramatic shift from focusing on volume (i.e., size of the target clinical population) to focusing on value (i.e., drug performance in real-world settings). In this context, value-based contracts (VBCs) were designed to align the payment of a drug to its clinical performance outside clinical trials by evaluating the effectiveness using real-word evidence (RWE). Despite their widespread implementation, different factors jeopardize the application of VBCs to most marketed drugs in a near future, including the need for easily measurable and relevant outcomes associated with clinical improvements, and access to a large patient population to assess said outcomes. Here, we argue that the extraction and analysis of massive amounts of RWE captured in patients' electronic health records (EHRs) will circumvent these issues and optimize negotiations in VBCs. Particularly, the use of Natural Language Processing (NLP) has proven successful in the analysis of structured and unstructured clinical information in EHRs in multicenter research studies. Thus, the application of NLP to analyze patient-centered information in EHRs in the context of innovative contracting can be utterly beneficial as it enables the real-time evaluation of treatment response and financial impact in real-world settings.
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BACKGROUND: The management of clinical heart failure with a preserved ejection fraction (HFpEF) is often complicated by concurrent renal dysfunction, known as the cardiorenal syndrome. This, combined with the notable lack of evidence-based therapies for HFpEF, highlights the importance of examining mechanisms and targetable pathways in HFpEF with the cardiorenal syndrome. METHODS: HFpEF was induced in mice by uninephrectomy, infusion of d-aldosterone (HFpEF; N=10) or saline (Sham; N=8), and given 1% NaCl drinking water for 4 weeks. Renal fibrosis and endothelial-mesenchymal transition (endo-MT) were evident once HFpEF developed. Human aortic endothelial cells were treated for 4 days with 10% serum obtained from patients with chronically stable HFpEF with the cardiorenal syndrome (N=12) and compared with serum-treated human aortic endothelial cells from control subjects (no cardiac/renal disease; N=12) to recapitulate the in vivo findings. RESULTS: Kidneys from HFpEF mice demonstrated hypertrophy, interstitial fibrosis (1.9-fold increase; P<0.05) with increased expression of endo-MT transcripts, including pdgfrß (platelet-derived growth factor receptor ß), snail, fibronectin, fsp1 (fibroblast-specific protein 1), and vimentin by 1.7- (P=0.004), 1.7- (P=0.05), 1.8- (P=0.005), 2.6- (P=0.001), and 2.0-fold (P=0.001) versus Sham. Immunostaining demonstrated co-localization of CD31 and ACTA2 (actin α2) in kidney sections suggesting evidence of endo-MT. Similar to the findings in HFpEF mice, comparable endo-MT markers were also significantly elevated in human aortic endothelial cells treated with serum from patients with HFpEF compared with human aortic endothelial cells treated with serum from control subjects. CONCLUSIONS: These translational findings demonstrate a plausible role for endo-MT in HFpEF with cardiorenal syndrome and may have therapeutic implications in drug development for patients with HFpEF and concomitant renal dysfunction.
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Síndrome Cardiorrenal/fisiopatologia , Células Endoteliais/metabolismo , Insuficiência Cardíaca/fisiopatologia , Volume Sistólico/fisiologia , Aldosterona/metabolismo , Biomarcadores/metabolismo , Síndrome Cardiorrenal/metabolismo , Humanos , Miocárdio/patologiaRESUMO
Background Posttranslational protein modification with O-linked N-acetylglucosamine (O-GlcNAc) is linked to high glucose levels in type 2 diabetes mellitus (T2DM) and may alter cellular function. We sought to elucidate the involvement of O-GlcNAc modification in endothelial dysfunction in patients with T2DM. Methods and Results Freshly isolated endothelial cells obtained by J-wire biopsy from a forearm vein of patients with T2DM (n=18) was compared with controls (n=10). Endothelial O-GlcNAc levels were 1.8-ford higher in T2DM patients than in nondiabetic controls (P=0.003). Higher endothelial O-GlcNAc levels correlated with serum fasting blood glucose level (r=0.433, P=0.024) and hemoglobin A1c (r=0.418, P=0.042). In endothelial cells from patients with T2DM, normal glucose conditions (24 hours at 5 mmol/L) lowered O-GlcNAc levels and restored insulin-mediated activation of endothelial nitric oxide synthase, whereas high glucose conditions (30 mmol/L) maintained both O-GlcNAc levels and impaired insulin action. Treatment of endothelial cells with Thiamet G, an O-GlcNAcase inhibitor, increased O-GlcNAc levels and blunted the improvement of insulin-mediated endothelial nitric oxide synthase phosphorylation by glucose normalization. Conclusions Taken together, our findings indicate a role for O-GlcNAc modification in the dynamic, glucose-induced impairment of endothelial nitric oxide synthase activation in endothelial cells from patients with T2DM. O-GlcNAc protein modification may be a treatment target for vascular dysfunction in T2DM.
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Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliais/efeitos dos fármacos , Antebraço/irrigação sanguínea , Glucose/farmacologia , Glucose/toxicidade , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Adulto , Estudos de Casos e Controles , Células Cultivadas , Diabetes Mellitus Tipo 2/diagnóstico , Células Endoteliais/metabolismo , Feminino , Glicosilação , Humanos , Insulina/farmacologia , Masculino , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo III/metabolismo , Fenótipo , Fosforilação , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
BACKGROUND: Immunoglobulin light chain (AL) cardiac amyloidosis is characterized by extracellular deposition of amyloid fibrils in the heart and is potentially fatal. Untreated, it manifests clinically as heart failure with a precipitous decline and a median survival of <6â¯months. AL cardiac amyloidosis is associated with impaired extracellular matrix homeostasis in the heart with increased matrix metalloproteinase (MMP) levels. This commmunication provides novel insights into a potential role for doxycycline, a non-selective MMP inhibitor in AL cardiac amyloidosis. METHODS/RESULTS: Adult rat ventricular myocytes stimulated with AL (obtained from cardiac amyloidosis patients) increased MMP-2 and MMP-9 activities (Pâ¯<â¯.05); the expression of autophagy marker microtubule associated protein 1 LC-3 isoform II (LC3-II) (Pâ¯<â¯.01), and the autophagy-related proteins ATG-4B (Pâ¯<â¯.05) and ATG-5 (P <â¯.05) as compared to untreated cardiomyocytes. Doxycycline abrogated MMP activities (Pâ¯<â¯.0001) and decreased AL-induced autophagy via ATG-5 (Pâ¯<â¯.05). CONCLUSIONS: These in vitro studies demonstrated that doxycycline, in addition to inhibiting MMP, also modulated AL-induced autophagy in cardiomyocytes and provide potential insights for future therapeutic targets for AL-induced proteotoxicity. Novel therapies for cardiotoxicity and heart failure in AL cardiac amyloidosis remain an important unmet need.
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Amiloidose , Miócitos Cardíacos , Animais , Autofagia , Doxiciclina/farmacologia , Humanos , Cadeias Leves de Imunoglobulina , Miocárdio , RatosRESUMO
Protein S-nitrosylation is a reversible post-translational modification of protein cysteines that is increasingly being considered as a signal transduction mechanism. The "biotin switch" technique marked the beginning of the study of the S-nitrosoproteome, based on the specific replacement of the labile S-nitrosylation by a more stable biotinylation that allowed further detection and purification. However, its application for proteomic studies is limited by its relatively low sensitivity. Thus, typical proteomic experiments require high quantities of protein extracts, which precludes the use of this method in a number of biological settings. We have developed a "fluorescence switch" technique that, when coupled to 2-DE proteomic methodologies, allows the detection and identification of S-nitrosylated proteins by using limited amounts of starting material, thus significantly improving the sensitivity. We have applied this methodology to detect proteins that become S-nitrosylated in endothelial cells when exposed to S-nitroso-L-cysteine, a physiological S-nitrosothiol, identifying already known S-nitrosylation targets, as well as proteins that are novel targets. This "fluorescence switch" approach also allowed us to identify several proteins that are denitrosylated by thioredoxin in cytokine-activated RAW264.7 (murine macrophage) cells. We believe that this method represents an improvement in order to approach the identification of S-nitrosylated proteins in physiological conditions.
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Cisteína/análogos & derivados , Processamento de Proteína Pós-Traducional , Proteínas/análise , Proteínas/metabolismo , Proteômica/métodos , S-Nitrosotióis/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Cisteína/análise , Cisteína/metabolismo , Células Endoteliais/citologia , Fluorescência , Humanos , Macrófagos/citologia , Camundongos , Nitrosação , S-Nitrosotióis/análise , Sensibilidade e EspecificidadeRESUMO
Background Prior studies have shown that nutrient excess induces endoplasmic reticulum ( ER ) stress in nonvascular tissues from patients with diabetes mellitus ( DM ). ER stress and the subsequent unfolded protein response may be protective, but sustained activation may drive vascular injury. Whether ER stress contributes to endothelial dysfunction in patients with DM remains unknown. Methods and Results To characterize vascular ER stress, we isolated endothelial cells from 42 patients with DM and 37 subjects without DM. Endothelial cells from patients with DM displayed higher levels of ER stress markers compared with controls without DM. Both the early adaptive response, evidenced by higher phosphorylated protein kinase-like ER eukaryotic initiation factor-2a kinase and inositol-requiring ER-to-nucleus signaling protein 1 ( P=0.02, P=0.007, respectively), and the chronic ER stress response evidenced by higher C/ EBP α-homologous protein ( P=0.02), were activated in patients with DM . Higher inositol-requiring ER-to-nucleus signaling protein 1 activation was associated with lower flow-mediated dilation, consistent with endothelial dysfunction ( r=0.53, P=0.02). Acute treatment with liraglutide, a glucagon-like peptide 1 receptor agonist, reduced p-inositol-requiring ER-to-nucleus signaling protein 1 ( P=0.01), and the activation of its downstream target c-jun N-terminal kinase ( P=0.025) in endothelial cells from patients with DM . Furthermore, liraglutide restored insulin-stimulated endothelial nitric oxide synthase activation in patients with DM ( P=0.019). Conclusions In summary, our data suggest that ER stress contributes to vascular insulin resistance and endothelial dysfunction in patients with DM . Further, we have demonstrated that liraglutide ameliorates ER stress, decreases c-jun N-terminal kinase activation and restores insulin-mediated endothelial nitric oxide synthase activation in endothelial cells from patients with DM .
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Diabetes Mellitus/tratamento farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Resistência à Insulina/fisiologia , Insulina/sangue , Liraglutida/farmacologia , Vasodilatação/fisiologia , Artéria Braquial/diagnóstico por imagem , Artéria Braquial/fisiopatologia , Células Cultivadas , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Feminino , Humanos , Hipoglicemiantes/farmacologia , Masculino , Pessoa de Meia-Idade , Ultrassonografia DopplerRESUMO
The accumulation of visceral adiposity is strongly associated with systemic inflammation and increased cardiometabolic risk. WNT5A, a non-canonical WNT ligand, has been shown to promote adipose tissue inflammation and insulin resistance in animal studies. Among other non-canonical pathways, WNT5A activates planar cell polarity (PCP) signaling. The current study investigated the potential contribution of non-canonical WNT5A/PCP signaling to visceral adipose tissue (VAT) inflammation and associated metabolic dysfunction in individuals with obesity. VAT and subcutaneous adipose tissue (SAT) samples obtained from subjects undergoing bariatric surgery were analyzed by qRT-PCR for expression of WNT/PCP genes. In vitro experiments were conducted with preadipocytes isolated from VAT and SAT biopsies. The expression of 23 out of 33 PCP genes was enriched in VAT compared to SAT. Strong positive expression correlations of individual PCP genes were observed in VAT. WNT5A expression in VAT, but not in SAT, correlated with indexes of JNK signaling activity, IL6, waist-to-hip ratio and hsCRP. In vitro, WNT5A promoted the expression of IL6 in human preadipocytes. In conclusion, elevated non-canonical WNT5A signaling in VAT contributes to the exacerbated IL-6 production in this depot and the low-grade systemic inflammation typically associated with visceral adiposity.
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Regulação da Expressão Gênica , Paniculite/metabolismo , Gordura Subcutânea/metabolismo , Via de Sinalização Wnt , Adulto , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Paniculite/patologia , Gordura Subcutânea/patologiaRESUMO
BACKGROUND: Endoplasmic reticulum (ER) stress and the subsequent unfolded protein response may initially be protective, but when prolonged, have been implicated in atherogenesis in diabetic conditions. Triglycerides and free fatty acids (FFAs) are elevated in patients with diabetes and may contribute to ER stress. We sought to evaluate the effect of acute FFA elevation on ER stress in endothelial and circulating white cells. METHODS AND RESULTS: Twenty-one healthy subjects were treated with intralipid (20%; 45 mL/h) plus heparin (12 U/kg/h) infusion for 5 hours. Along with increased triglyceride and FFA levels, intralipid/heparin infusion reduced the calf reactive hyperemic response without a change in conduit artery flow-mediated dilation consistent with microvascular dysfunction. To investigate the short-term effects of elevated triglycerides and FFA, we measured markers of ER stress in peripheral blood mononuclear cells (PBMCs) and vascular endothelial cells (VECs). In VECs, activating transcription factor 6 (ATF6) and phospho-inositol requiring kinase 1 (pIRE1) proteins were elevated after infusion (both P<0.05). In PBMCs, ATF6 and spliced X-box-binding protein 1 (XBP-1) gene expression increased by 2.0- and 2.5-fold, respectively (both P<0.05), whereas CHOP and GADD34 decreased by ≈67% and 74%, respectively (both P<0.01). ATF6 and pIRE1 protein levels also increased (both P<0.05), and confocal microscopy revealed the nuclear localization of ATF6 after infusion, suggesting activation. CONCLUSIONS: Along with microvascular dysfunction, intralipid infusion induced an early protective ER stress response evidenced by activation of ATF6 and IRE1 in both leukocytes and endothelial cells. Our results suggest a potential link between metabolic disturbances and ER stress that may be relevant to vascular disease.
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Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Perna (Membro)/irrigação sanguínea , Leucócitos Mononucleares/efeitos dos fármacos , Fosfolipídeos/administração & dosagem , Óleo de Soja/administração & dosagem , Fator 6 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/metabolismo , Adulto , Anticoagulantes/administração & dosagem , Biomarcadores/metabolismo , Emulsões/administração & dosagem , Endorribonucleases/metabolismo , Células Endoteliais/metabolismo , Feminino , Regulação da Expressão Gênica , Voluntários Saudáveis , Heparina/administração & dosagem , Humanos , Hiperemia/fisiopatologia , Infusões Intravenosas , Leucócitos Mononucleares/metabolismo , Masculino , Microcirculação/efeitos dos fármacos , Fosfolipídeos/sangue , Fosforilação , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Óleo de Soja/sangue , Fatores de Tempo , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo , Adulto JovemRESUMO
BACKGROUND: Endothelial dysfunction contributes to cardiovascular disease in diabetes mellitus. Autophagy is a multistep mechanism for the removal of damaged proteins and organelles from the cell. Under diabetic conditions, inadequate autophagy promotes cellular dysfunction and insulin resistance in non-vascular tissue. We hypothesized that impaired autophagy contributes to endothelial dysfunction in diabetes mellitus. METHODS AND RESULTS: We measured autophagy markers and endothelial nitric oxide synthase (eNOS) activation in freshly isolated endothelial cells from diabetic subjects (n = 45) and non-diabetic controls (n = 41). p62 levels were higher in cells from diabetics (34.2 ± 3.6 vs. 20.0 ± 1.6, P = 0.001), indicating reduced autophagic flux. Bafilomycin inhibited insulin-induced activation of eNOS (64.7 ± 22% to -47.8 ± 8%, P = 0.04) in cells from controls, confirming that intact autophagy is necessary for eNOS signaling. In endothelial cells from diabetics, activation of autophagy with spermidine restored eNOS activation, suggesting that impaired autophagy contributes to endothelial dysfunction (P = 0.01). Indicators of autophagy initiation including the number of LC3-bound puncta and beclin 1 expression were similar in diabetics and controls, whereas an autophagy terminal phase indicator, the lysosomal protein Lamp2a, was higher in diabetics. In endothelial cells under diabetic conditions, the beneficial effect of spermidine on eNOS activation was blocked by autophagy inhibitors bafilomycin or 3-methyladenine. Blocking the terminal stage of autophagy with bafilomycin increased p62 (P = 0.01) in cells from diabetics to a lesser extent than in cells from controls (P = 0.04), suggesting ongoing, but inadequate autophagic clearance. CONCLUSION: Inadequate autophagy contributes to endothelial dysfunction in patients with diabetes and may be a target for therapy of diabetic vascular disease.
Assuntos
Autofagia/efeitos dos fármacos , Diabetes Mellitus Tipo 2/patologia , Angiopatias Diabéticas/patologia , Células Endoteliais/efeitos dos fármacos , Óxido Nítrico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espermidina/farmacologia , Adenosina/análogos & derivados , Adenosina/farmacologia , Adulto , Idoso , Biomarcadores/metabolismo , Estudos de Casos e Controles , Separação Celular/métodos , Células Cultivadas , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/fisiopatologia , Angiopatias Diabéticas/sangue , Angiopatias Diabéticas/fisiopatologia , Angiopatias Diabéticas/prevenção & controle , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Humanos , Macrolídeos/farmacologia , Masculino , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo III/metabolismoRESUMO
Laminar shear stress (LSS) triggers signals that ultimately result in atheroprotection and vasodilatation. Early responses are related to the activation of specific signaling cascades. We investigated the participation of redox-mediated modifications and in particular the role of hydrogen peroxide (H2O2) in the sulfenylation of redox-sensitive phosphatases. Exposure of vascular endothelial cells to short periods of LSS (12 dyn/cm(2)) resulted in the generation of superoxide radical anion as detected by the formation of 2-hydroxyethidium by HPLC and its subsequent conversion to H2O2, which was corroborated by the increase in the fluorescence of the specific peroxide sensor HyPer. By using biotinylated dimedone we detected increased total protein sulfenylation in the bovine proteome, which was dependent on NADPH oxidase 4 (NOX4)-mediated generation of peroxide. Mass spectrometry analysis allowed us to identify the phosphatase SHP2 as a protein susceptible to sulfenylation under LSS. Given the dependence of FAK activity on SHP2 function, we explored the role of FAK under LSS conditions. FAK activation and subsequent endothelial NO synthase (eNOS) phosphorylation were promoted by LSS and both processes were dependent on NOX4, as demonstrated in lung endothelial cells isolated from NOX4-null mice. These results support the idea that LSS elicits redox-sensitive signal transduction responses involving NOX4-dependent generation of hydrogen peroxide, SHP2 sulfenylation, and ulterior FAK-mediated eNOS activation.
Assuntos
Peróxido de Hidrogênio/farmacologia , NADPH Oxidases/fisiologia , Óxido Nítrico Sintase Tipo III/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/química , Estresse Mecânico , Ácidos Sulfênicos/química , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/patologia , Western Blotting , Bovinos , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Feminino , Imunofluorescência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 4 , Óxido Nítrico/metabolismo , Oxidantes/farmacologia , Oxirredução , Fosforilação/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Transdução de Sinais/efeitos dos fármacos , SuperóxidosRESUMO
Redox signaling is implicated in different physiological and pathological events in the vasculature. Among the different reactive oxygen species, hydrogen peroxide (H2O2) is a very good candidate to perform functions as an intracellular messenger in the regulation of several biological events. In this review, we summarize the main physiological sources of H2O2 in the endothelium and the molecular mechanisms by which it is able to act as a signaling mediator in the vasculature.
Assuntos
Células Endoteliais/metabolismo , Peróxido de Hidrogênio/metabolismo , Oxirredutases/metabolismo , Transdução de Sinais/fisiologia , Vasodilatação/fisiologia , Animais , Doenças Cardiovasculares/metabolismo , Divisão Celular , Hemorreologia , Humanos , Mitocôndrias/metabolismo , Modelos Biológicos , NADPH Oxidases/metabolismo , Neovascularização Fisiológica/fisiologia , Óxido Nítrico Sintase Tipo III/metabolismo , Oxirredução , Estresse Oxidativo , Proteínas Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Xantina Desidrogenase/metabolismoRESUMO
Substantial evidence suggests that a transient increase of hydrogen peroxide (H2O2) behaves as an intracellular messenger able to trigger the activation of different signaling pathways. These include phosphatases, protein kinases, and transcription factors among others; however, most of the studies have been performed using supraphysiological levels of H2O2. Reactive oxygen species (ROS) generation occurs under physiological conditions and different extracellular stimuli including cytokines, growth factors, and shear stress are able to produce both low levels of superoxide anion and H2O2. Here, we explore the redox-dependent activation of key signaling pathways induced by shear stress. We demonstrate that laminar shear stress (LSS) rapidly promotes a transient generation of H2O2 that is necessary for the activation of the stress-activated protein kinase p38 MAPK. We describe p38 MAPK as an early redox sensor in LSS. Our studies show that it is essential for the activation of endothelial nitric oxide synthase, the subsequent nitric oxide generation, and the protection of endothelial function.