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BACKGROUND: Genome-wide association studies have discovered a link between genetic variants on human chromosome 15q26.1 and increased coronary artery disease (CAD) susceptibility; however, the underlying pathobiological mechanism is unclear. This genetic locus contains the FES (FES proto-oncogene, tyrosine kinase) gene encoding a cytoplasmic protein-tyrosine kinase involved in the regulation of cell behavior. We investigated the effect of the 15q26.1 variants on FES expression and whether FES plays a role in atherosclerosis. METHODS AND RESULTS: Analyses of isogenic monocytic cell lines generated by CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing showed that monocytes with an engineered 15q26.1 CAD risk genotype had reduced FES expression. Small-interfering-RNA-mediated knockdown of FES promoted migration of monocytes and vascular smooth muscle cells. A phosphoproteomics analysis showed that FES knockdown altered phosphorylation of a number of proteins known to regulate cell migration. Single-cell RNA-sequencing revealed that in human atherosclerotic plaques, cells that expressed FES were predominately monocytes/macrophages, although several other cell types including smooth muscle cells also expressed FES. There was an association between the 15q26.1 CAD risk genotype and greater numbers of monocytes/macrophage in human atherosclerotic plaques. An animal model study demonstrated that Fes knockout increased atherosclerotic plaque size and within-plaque content of monocytes/macrophages and smooth muscle cells, in apolipoprotein E-deficient mice fed a high fat diet. CONCLUSIONS: We provide substantial evidence that the CAD risk variants at the 15q26.1 locus reduce FES expression in monocytes and that FES depletion results in larger atherosclerotic plaques with more monocytes/macrophages and smooth muscle cells. This study is the first demonstration that FES plays a protective role against atherosclerosis and suggests that enhancing FES activity could be a potentially novel therapeutic approach for CAD intervention.
Assuntos
Aterosclerose , Doença da Artéria Coronariana , Placa Aterosclerótica , Proteínas Proto-Oncogênicas c-fes , Animais , Humanos , Camundongos , Artérias/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/metabolismo , Estudo de Associação Genômica Ampla , Miócitos de Músculo Liso/metabolismo , Placa Aterosclerótica/genética , Placa Aterosclerótica/metabolismo , Proteínas Proto-Oncogênicas c-fes/genética , Proteínas Proto-Oncogênicas c-fes/metabolismoRESUMO
BACKGROUND: Thoracic aortic dissection (TAD) is a life-threatening aortic disease without effective medical treatment. Increasing evidence has suggested a role for NE (neutrophil elastase) in vascular diseases. In this study, we aimed at investigating a causal role for NE in TAD and exploring the molecular mechanisms involved. METHODS: ß-aminopropionitrile monofumarate was administrated in mice to induce TAD. NE deficiency mice, pharmacological inhibitor GW311616A, and adeno-associated virus-2-mediated in vivo gene transfer were applied to explore a causal role for NE and associated target gene in TAD formation. Multiple functional assays and biochemical analyses were conducted to unravel the underlying cellular and molecular mechanisms of NE in TAD. RESULTS: NE aortic gene expression and plasma activity was significantly increased during ß-aminopropionitrile monofumarate-induced TAD and in patients with acute TAD. NE deficiency prevents ß-aminopropionitrile monofumarate-induced TAD onset/development, and GW311616A administration ameliorated TAD formation/progression. Decreased levels of neutrophil extracellular traps, inflammatory cells, and MMP (matrix metalloproteinase)-2/9 were observed in NE-deficient mice. TBL1x (F-box-like/WD repeat-containing protein TBL1x) has been identified as a novel substrate and functional downstream target of NE in TAD. Loss-of-function studies revealed that NE mediated inflammatory cell transendothelial migration by modulating TBL1x-LTA4H (leukotriene A4 hydrolase) signaling and that NE regulated smooth muscle cell phenotype modulation under TAD pathological condition by regulating TBL1x-MECP2 (methyl CpG-binding protein 2) signal axis. Further mechanistic studies showed that TBL1x inhibition decreased the binding of TBL1x and HDAC3 (histone deacetylase 3) to MECP2 and LTA4H gene promoters, respectively. Finally, adeno-associated virus-2-mediated Tbl1x gene knockdown in aortic smooth muscle cells confirmed a regulatory role for TBL1x in NE-mediated TAD formation. CONCLUSIONS: We unravel a critical role of NE and its target TBL1x in regulating inflammatory cell migration and smooth muscle cell phenotype modulation in the context of TAD. Our findings suggest that the NE-TBL1x signal axis represents a valuable therapeutic for treating high-risk TAD patients.
Assuntos
Aneurisma da Aorta Torácica , Dissecção Aórtica , Dissecção da Aorta Torácica , Animais , Humanos , Camundongos , Aminopropionitrilo/toxicidade , Aneurisma da Aorta Torácica/induzido quimicamente , Aneurisma da Aorta Torácica/genética , Aneurisma da Aorta Torácica/metabolismo , Dissecção Aórtica/induzido quimicamente , Dissecção Aórtica/genética , Elastase de Leucócito/genética , Elastase de Leucócito/efeitos adversosRESUMO
IMP3, an RNA-binding protein (RBP) that participates in the process of post-transcriptional modifications of mRNA transcripts, is capable of altering cellular functions, and in some cases, be involved in specific disease progression. We aimed to investigate whether IMP3 has the ability to regulate the functional properties of endothelial cells and re-endothelialization in response to arterial injury. Wire injury was introduced to the right carotid arteries of wildtype C57/BL6 mice. As a result, IMPs' expressions were up-regulated in the induced arterial lesions, and IMP3 was the most up-regulated RNA among other IMPs. We overexpressed IMP3 before the wire-injured surgery using adeno-associated virus AAV2-IMP3. In vivo studies confirmed that IMP3 overexpression accelerated the progress of re-endothelialization after arterial injury. In vitro, endothelial cells were transfected with either ad-IMP3 or Si-IMP3, cell functional studies showed that IMP3 could promote endothelial cell proliferation and migration, while reducing apoptosis. Mechanistic studies also revealed that IMP3 could enhance VEGF mRNA stability and therefore up-regulate activities of VEGF/PI3K/Akt signalling pathway. Our data indicated that IMP3 promotes re-endothelialization after arterial injury and regulates endothelial cell proliferation, migration and apoptosis via increasing stability of VEGF mRNA and activation of VEGF/PI3K/Akt signalling pathway.
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Células Endoteliais , Proteínas de Ligação a RNA , Lesões do Sistema Vascular , Animais , Proliferação de Células/genética , Células Endoteliais/patologia , Camundongos , Fosfatidilinositol 3-Quinases , Proteínas de Ligação a RNA/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Lesões do Sistema Vascular/patologiaRESUMO
Forecasting the value at risk (VaR) of crude oil futures can be a challenging task for investors due to the high volatility of these prices. It is crucial to describe the return in the tail distribution, as extreme values can trigger larger price fluctuations and market risks. In this study, we proposed a hybrid model, ARIMA-SVR-POT, which uses a combination of the autoregressive integrated moving average (ARIMA), support vector regression (SVR), and peak over threshold (POT) method from the extreme value theory. We compared the performance of our hybrid model with three other models, namely ARIMA-EGARCH, ARIMA-SVR, and ARIMA-EGARCH-POT. We demonstrated the effectiveness of our model using crude oil WTI Futures as a sample from June 23, 2016, to September 30, 2022. Our findings show that the ARIMA-SVR-POT hybrid model provides accurate predictions of the returns and volatility. Furthermore, the model performs exceptionally well in capturing the extreme tail of returns and outperforms the other models. We also conducted back-testing in the proposed model and the results show that the ARIMA-SVR-POT model passed the Kupiec test at confidence levels of 95 %, 99 %, 99.5 %, and 99.9 %. Our proposed model provides a more precise reflection of potential losses when estimating VaR. The predicted loss probability is closer to the actual loss occurrence probability, indicating superior performance compared to traditional statistical models, which enhanced crude oil risk management tools and suggested effective measures to manage market risks.
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Green funds play pivotal roles in driving corporate sustainable development. Utilizing data from Chinese publicly listed companies from 2010 to 2021, we examine the impact of green funds on corporate environmental, social, and governance (ESG) performance and the underlying mechanisms. The research findings claim that green funds positively affect corporate ESG performance. Mechanism analysis systematically demonstrates that green funds contribute to elevated corporate ESG performance by alleviating financial constraints, enhancing managerial efficiency, and fostering green innovation. Heterogeneity analysis further underscores that the effect of green funds is particularly potent in companies with high external attention. Furthermore, green funds also play significant roles in production capabilities and economic value. This research enriches the micro-level evidence on the development of green funds and furnishes substantial implications for sustainable development.
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The issue of misjudgment in recombinant erythropoietin (rEPO) detection caused by the variant c.577del in human EPO gene has been found in recent years. Though the method of analyzing de-N-glycosylated erythropoietins (EPO) in blood samples was developed for identifying both EPO_p.Arg193AspfsTer28 (VAR-EPO) and rEPO, it cannot be applied without the evaluation of excreted samples. For this purpose, five heterozygous carriers of the variant c.577del were recruited in an administration study of rEPO. Urine and blood samples were collected at different times before and after subcutaneous injection with a single-dose of 50 IU/kg. The urine samples were analyzed for intact EPO, while the serum samples were analyzed for both intact and de-N-glycosylated EPO. A typical mixed band was detected in all blank and wash-out urine samples, which all displayed a similar result with rEPO abuse. For the analysis of intact EPO in serum samples, a typical mixed band was detected in the wash-out samples from day 1 to day 3, which could be identified as rEPO directly, while double-band was observed in other samples with inconclusive results. The result of de-N-glycosylated EPO in all serum samples showed two separated bands, and the ratioL/U decreased along with wash-out periods. Also, compared with the intact EPO analysis, a longer detection window without false positive results was obtained when analyzing de-N-glycosylated EPO. Analysis of de-N-glycosylated EPO is not only able to recognize the variant carriers directly but also able to detect rEPO abuse in the blood samples from the variant carriers with higher efficiency than the analysis of intact EPO.
Assuntos
Eritropoetina , Humanos , Proteínas RecombinantesRESUMO
The abuse of recombinant erythropoietin (rEPO) and other erythropoietin (EPO) receptor agonists (ERAs) in sports prompted the need for sensitive detection methods of these substances. Dried blood spot (DBS) samples offer an easy solution for simultaneous collection of blood and urine during a doping control, but sensitivity issues are often presented as a challenge for routine EPO analysis from DBS. Its potential use for detecting rEPO micro-doses and the EPO gene c.577del variant thus needed further demonstration. Here, capillary blood collected from the arm skin of 111 athletes with Tasso-M20 (17.5 µL/spot), collected during professional triathlon competitions, were analysed. Also, venous blood samples from healthy volunteers were used to prepare several spots of 20 µL on Mitra VAMS (from an rEPO micro-dose study) and Whatman filter paper (from an EPO gene variant study). Immunopurification of 2 spots with MAIIA EPO Purification Gel Kit and analysis with sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SAR-PAGE)/Western blot resulted in sensitive detection of (1) micro-doses of rEPO from Mitra VAMS, (2) endogenous EPO from Tasso-M20 in all in-competition subjects, and (3) the EPO c.577del variant from Whatman filter paper. Additionally, in-competition endogenous EPO was detected in DBS even when matching urine samples had undetectable EPO. In conclusion, this work demonstrated that DBS can be a useful complementary matrix to urine samples for EPO detection.
RESUMO
Frameshift variant c.577del in the EPO gene can result in the extension of the amino acid sequence of EPO by invalidating the termination codon. As the molecular weight of its encoded protein EPO (VAR-EPO) is similar to that of rEPO, the World Anti-Doping Agency has published Annex B to the TD2022EPO in order to protect athletes with variant c.577del from the suspicion of rEPO administrations. However, it is still necessary to develop a confirmation method for rEPO that can discriminate rEPO from VAR-EPO. Based on the glycosylated characteristic of EPO, we selected the detection of de-N-glycosylated EPO as a complementary confirmation method for rEPO in blood samples. All samples were analyzed for both intact EPO and de-N-glycosylated EPO with SDS-PAGE, including rEPO spiked samples and blank samples. The results showed that, after de-N-glycosylation, a single-band was detected in samples collected from non-variant carriers, no matter whether the sample was spiked with rEPO. In samples collected from variant carriers, a double-band was detected. The ratio of lower band to upper band increased significantly corresponding to the concentration of rEPO. We calculated a series of cut-off values by normality distribution function to identify the presence of rEPO. Neither false positive results in blank samples nor false negative results in spiked samples at the applicable Minimum Required Performance Levels were found. This indicates that this method could be adopted as a complementary confirmation method for rEPO in blood samples. A revised testing strategy was also proposed, which would discriminate rEPO directly without further investigation.
Assuntos
Eritropoetina , Humanos , Glicosilação , Detecção do Abuso de Substâncias/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eletroforese em Gel de PoliacrilamidaRESUMO
As a mixed-methods research in economics and psychology, this study aimed to analyze the influence from the intergenerational succession on the financialization level including asset financialization and revenue financialization, and further test the moderating effect of the heirs' typical growing experience according to The Imprinting Theory, based on the 2009-2020 annual data of listed family enterprises of China. There were two key findings. First, the effect of Chinese family enterprises' intergenerational succession on asset financialization was positively significant while the effect on revenue financialization was not significant, indicating that the financialization behavior has not brought about effective financial profits. Second, among the heirs' typical growing experiences, their parents' entrepreneurial experience during their childhood, oversea study experience, and MBA education experience had the significantly positive moderating effects on the influence from intergeneration succession to asset financialization level of Chinese family enterprises, which was an important internal mechanism for the heirs to promote the financialization process of family enterprises.
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Recently, some athletes were repetitively found to have rEPO positive results, including a characterized double-band pattern in blood samples, in routine doping analysis. In contrast to previous findings from excretion studies, this double-band pattern showed the same relative intensity even when the samples were collected weeks (/months) apart. We therefore suspected that these "positive" doping control samples were related with a novel pathway of endogenous EPO production. Thus, follow-up investigations were warranted to characterize the origin of such analytical test results and to avoid the issuing of adverse analytical findings in the absence of rEPO by identifying the root cause of these "constantly positives." In this study, we designed and conducted a series of causal studies, including population screening of EPO profiles, exploration of EPO de-N-glycosylation, single nucleotide polymorphism (SNP) browsing in EPO, sequencing of EPO exons, genealogical analysis of the c.577del EPO variant, and finally expression and investigation of mutant EPO. In summary, we found that these "constantly positives" were related to endogenous EPO production associated with the c.577del EPO variant. The frequency of this variant was 0.39% in our Chinese population pool. The mutant EPO encoded by this variant is 27 amino acids longer than the wild-type. The molecular weight of this mutant EPO is approximately the same as that of rEPO, exhibiting a similar electrophoretic behavior. To prevent charges against carriers of the c.577del variant, a revised rEPO testing strategy has been implemented in the new version of TD EPO.
Assuntos
Dopagem Esportivo , Eritropoetina , Dopagem Esportivo/prevenção & controle , Humanos , Proteínas Recombinantes/genética , Detecção do Abuso de Substâncias/métodosRESUMO
Jimaixin™ (Jintan Ltd, China) is a biosimilar of recombinant erythropoietin (rEPO) now authorized for therapeutic application in China. With a risk of abuse by athletes, a clear evaluation of its detection using the electrophoretic methods in use in antidoping laboratories was necessary. In a previous work, we showed that Jimaixin™ electrophoretic profile presented slight changes compared with the original drug (first generation rEPO) and that a spike of Jimaixin™ in urine and serum was well identified by SDS-PAGE but with less performance by IEF-PAGE unless a neuraminidase treatment was applied first. The aims of this research were to perform an intravenous administration of Jimaixin™ on three healthy subjects (one microdose [10 IU/kg] and three therapeutic doses [50 IU/kg]) and to evaluate the detection in urine and blood up to 7 days post administration. Analysis of the samples showed that Jimaixin™ detection was complicated by IEF-PAGE due to the loss of the most distinctive basic isoforms. In addition, a neuraminidase treatment did not improve detection (contrary to the observations from spike experiments). On the contrary, Jimaixin™ was very efficiently detected in blood and urine by SDS-PAGE: up to 40 h after a microdose and up to 7 days after the therapeutic doses. The effect of Jimaixin™ on hematological parameters was limited to a clear but transitory increase of the reticulocytes. These data give new elements to better survey a potential misuse of Jimaixin™ by athletes.
Assuntos
Medicamentos Biossimilares/análise , Dopagem Esportivo/prevenção & controle , Eritropoetina/análise , Detecção do Abuso de Substâncias/métodos , Administração Intravenosa , Adulto , Medicamentos Biossimilares/administração & dosagem , Medicamentos Biossimilares/farmacocinética , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida/métodos , Eritropoetina/administração & dosagem , Eritropoetina/farmacocinética , Humanos , Masculino , Proteínas Recombinantes , Fatores de TempoRESUMO
Vascular smooth muscle cells (VSMCs) play a central role in the progression of atherosclerosis, where they switch from a contractile to a synthetic phenotype. Because of their role as risk factors for atherosclerosis, we sought here to systematically study the impact of matrix stiffness and (hemodynamic) pressure on VSMCs. Thereby, we find that pressure and stiffness individually affect the VSMC phenotype. However, only the combination of hypertensive pressure and matrix compliance, and as such mechanical stimuli that are prevalent during atherosclerosis, leads to a full phenotypic switch including the formation of matrix-degrading podosomes. We further analyze the molecular mechanism in stiffness and pressure sensing and identify a regulation through different but overlapping pathways culminating in the regulation of the actin cytoskeleton through cofilin. Together, our data show how different pathological mechanical signals combined but through distinct pathways accelerate a phenotypic switch that will ultimately contribute to atherosclerotic disease progression.
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Aterosclerose , Músculo Liso Vascular , Aterosclerose/patologia , Proliferação de Células , Células Cultivadas , Humanos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , FenótipoRESUMO
Aortic dissection (AD) is a lethal aortic pathology without effective medical treatments since the underlying pathological mechanisms responsible for AD remain elusive. Matrix metalloproteinase-8 (MMP8) has been previously identified as a key player in atherosclerosis and arterial remodeling. However, the functional role of MMP8 in AD remains largely unknown. Here, we report that an increased level of MMP8 was observed in 3-aminopropionitrile fumarate (BAPN)-induced murine AD. AD incidence and aortic elastin fragmentation were markedly reduced in MMP8-knockout mice. Importantly, pharmacologic inhibition of MMP8 significantly reduced the AD incidence and aortic elastin fragmentation. We observed less inflammatory cell accumulation, a lower level of aortic inflammation, and decreased smooth muscle cell (SMC) apoptosis in MMP8-knockout mice. In line with our previous observation that MMP8 cleaves Ang I to generate Ang II, BAPN-treated MMP8-knockout mice had increased levels of Ang I, but decreased levels of Ang II and lower blood pressure. Additionally, we observed a decreased expression level of vascular cell adhesion molecule-1 (VCAM1) and a reduced level of reactive oxygen species (ROS) in MMP8-knockout aortas. Mechanistically, our data show that the Ang II/VCAM1 signal axis is responsible for MMP8-mediated inflammatory cell invasion and transendothelial migration, while MMP8-mediated SMC inflammation and apoptosis are attributed to Ang II/ROS signaling. Finally, we observed higher levels of aortic and serum MMP8 in patients with AD. We therefore provide new insights into the molecular mechanisms underlying AD and identify MMP8 as a potential therapeutic target for this life-threatening aortic disease.
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Dissecção Aórtica , Metaloproteinase 8 da Matriz , Animais , Camundongos , Aminopropionitrilo/farmacologia , Dissecção Aórtica/sangue , Dissecção Aórtica/genética , Angiotensina II/farmacologia , Angiotensina II/metabolismo , Modelos Animais de Doenças , Elastina/metabolismo , Inflamação/genética , Metaloproteinase 8 da Matriz/sangue , Metaloproteinase 8 da Matriz/genética , Metaloproteinase 8 da Matriz/metabolismo , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , HumanosRESUMO
AIMS: Pathological arterial remodelling including neointimal hyperplasia and atherosclerosis is the main underlying cause for occluding arterial diseases. Cezanne is a novel deubiquitinating enzyme, functioning as a NF-кB negative regulator, and plays a key role in renal inflammatory response and kidney injury induced by ischaemia. Here we attempted to examine its pathological role in vascular smooth muscle cell (VSMC) pathology and arterial remodelling. METHODS AND RESULTS: Cezanne expression levels were consistently induced by various atherogenic stimuli in VSMCs, and in remodelled arteries upon injury. Functionally, VSMCs over-expressing wild-type Cezanne, but not the mutated catalytically-inactive Cezanne (C209S), had an increased proliferative ability and mobility, while the opposite was observed in VSMCs with Cezanne knockdown. Surprisingly, we observed no significant effects of Cezanne on VSMC apoptosis, NF-κB signalling, or inflammation. RNA-sequencing and biochemical studies showed that Cezanne drives VSMC proliferation by regulating CCN family member 1 (CCN1) by targeting ß-catenin for deubiquitination. Importantly, local correction of Cezanne expression in the injured arteries greatly decreased VSMC proliferation, and prevented arterial inward remodelling. Interestingly, global Cezanne gene deletion in mice led to smaller atherosclerotic plaques, but with a lower level of plaque stability. Translating, we observed a similar role for Cezanne in human VSMCs, and higher expression levels of Cezanne in human atherosclerotic lesions. CONCLUSION: Cezanne is a key regulator of VSMC proliferation and migration in pathological arterial remodelling. Our findings have important implications for therapeutic targeting Cezanne signalling and VSMC pathology in vascular diseases.
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Aterosclerose/enzimologia , Endopeptidases/metabolismo , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Remodelação Vascular , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Apoptose , Aterosclerose/genética , Aterosclerose/patologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Proteína Rica em Cisteína 61/genética , Proteína Rica em Cisteína 61/metabolismo , Modelos Animais de Doenças , Endopeptidases/genética , Humanos , Mediadores da Inflamação/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , NF-kappa B/metabolismo , Neointima , Ubiquitinação , beta Catenina/genéticaRESUMO
BACKGROUND AND PURPOSE: Neointimal hyperplasia (NIH) is the fundamental cause for vascular diseases and vascular smooth muscle cell (VSMC) dysregulation has been widely implicated in NIH. Neutrophil elastase is a potential therapeutic target for multiple diseases. We investigated the role of neutrophil elastase in VSMC functions and injury-induced NIH and explored the therapeutic potential of targeting neutrophil elastase in NIH. EXPERIMENTAL APPROACH: VSMCs were used to analyse the effects of neutrophil elastase. Proteomic analysis was used to identify potential neutrophil elastase targets. Artery injury model and neutrophil elastase inhibitor GW311616A were used to investigate the role of neutrophil elastase in NIH. KEY RESULTS: TNF-α up-regulated neutrophil elastase in VSMCs through modulating GAPBα/Runx1/CEBPα/c-Myb signalling. Up-regulated neutrophil elastase promoted VSMC migration, proliferation and inflammation. Toll-like receptor 4 (TLR4) was identified as a target protein for neutrophil elastase in VSMCs and the TLR4/MyD88/IRAK1/TRAF6/NF-κB regulatory axis was shown to be the signalling pathway for neutrophil elastase in VSMC pathology. Importantly, TLR4 inhibition abolished neutrophil elastase-mediated VSMC dysregulation. Injury-induced NIH was significantly reduced in both neutrophil elastase-deficient mice and mice treated with GW311616A. The formation of neutrophil extracellular traps was impaired in injured arteries from neutrophil elastase-deficient mice. Finally, a similar role for neutrophil elastase in human VSMC pathology was confirmed and we observed higher expression levels of neutrophil elastase but lower expression levels of TLR4 in human atherosclerotic lesions. CONCLUSION AND IMPLICATIONS: We provide new insight into the molecular mechanisms underlying NIH and identify neutrophil elastase as a potential therapeutic target for vascular disease.
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NF-kappa B , Receptor 4 Toll-Like , Animais , Movimento Celular , Proliferação de Células , Células Cultivadas , Hiperplasia/patologia , Elastase de Leucócito , Camundongos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Neointima/patologia , ProteômicaRESUMO
According to the current Technical Document (TD) for erythropoietin (EPO), SAR-PAGE is the most commonly applied method for both screening and confirmation procedures. Although this method is effective and robust, it lacks an internal standard (IS) to monitor the efficiency of analysis for each sample covering every step of the whole procedure, including preparation, immunopurification, and western blotting. This internal standard needs to be recognized by both anti-EPO antibodies used for immunopurification and western blotting, respectively. Besides that, the band of IS could not be allowed to interfere with the recognition of all types of targeted EPO and analogs. To meet these two principles, rat EPO was selected. In this study, rat EPO was used to spike both urine and blood samples at the beginning of analysis. After preparation and immunopurification, single blotting was performed with biotinylated AE7A5 as the primary antibody, followed by incubation with streptavidin-coupled HRP. Based on the comparison of different immunopurification methods, the AB-286-NA antibody coupled to M-280 magnetic beads was the better choice for urine samples, whereas the MAIIA column was suitable for blood samples. All these methods were validated for selectivity, repeatability, and sensitivity. The modified method in this study could not only eliminate the cross-reactivity between antibodies but also monitor the whole procedure of the analysis of EPO with spiked rat EPO. Besides that, rat EPO could also be used as an indicator for monitoring the presence of protease(s) in urine samples.
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Dopagem Esportivo/prevenção & controle , Eletroforese em Gel de Poliacrilamida/métodos , Eritropoetina/análise , Detecção do Abuso de Substâncias/métodos , Animais , Anticorpos/imunologia , Biotinilação , Western Blotting , Eritropoetina/sangue , Eritropoetina/urina , Feminino , Humanos , Masculino , Ratos , Reprodutibilidade dos TestesRESUMO
Erythropoietins (EPOs) are substances listed in S2 of the World Anti-Doping Agency (WADA) Prohibited List and are used commonly by athletes to increase endurance performance. According to the current WADA Technical Documents, sarcosyl-polyacrylamide gel electrophoresis (SAR-PAGE) followed by western blotting to differentiate erythropoietins based on their molecular weights is the only method that can be used for both screening and confirmation of all types of erythropoietins. The efficiency of immunopurification and protein transfer is crucial for ensuring the selectivity and sensitivity of erythropoietin detection. Several comparisons and optimization of the SAR-PAGE tests were conducted in this study. We optimized the first blotting conditions and then compared different immunopurification methods based on their selectivity, repeatability, and sensitivity for both urine and blood analysis. Additionally, rapid procedures for both urine and blood analysis were established and compared. The two-step procedure at 1.0 mA/cm2 for 60 min followed by 1.56 mA/cm2 for 20 min increased the blotting efficiency compared with the commonly used constant current approach. Comparison of immunopurification revealed no significant difference in selectivity and sensitivity between the different methods. For other factors, such as operation complexity, time and cost, a StemCell® purification kit followed by single blotting and magnetic beads followed by double blotting are recommended for urine screening and confirmation, respectively. While magnetic beads and a MAIIA® kit followed by double blotting are recommended for both screening and confirmation of blood samples, respectively. To ensure high sensitivity and selectivity, double blotting is recommended for a rapid procedure for both urine and blood analysis.
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Eletroforese em Gel de Poliacrilamida/métodos , Eritropoetina/sangue , Eritropoetina/urina , Western Blotting/economia , Western Blotting/métodos , Dopagem Esportivo , Eletroforese em Gel de Poliacrilamida/economia , Humanos , Reprodutibilidade dos Testes , Detecção do Abuso de Substâncias/economia , Detecção do Abuso de Substâncias/métodos , Fatores de TempoRESUMO
BACKGROUND: Renal dysfunction is a serious morbidity in cirrhotic patients with acute upper gastrointestinal bleeding (AUGIB). Terlipressin is the first-line treatment choice for acute variceal bleeding and hepatorenal syndrome (HRS). This study aimed to assess the effect of terlipressin on renal function in patients with liver cirrhosis and AUGIB. METHODS: We retrospectively reviewed 40 cirrhotic patients with AUGIB treated with terlipressin by an attending physician between January 2016 and June 2018. We analyzed the change of renal function parameters, including cystatin C (CysC) and creatinine (Cr), during the use of terlipressin and after terlipressin was stopped. We also identified the factors associated with renal function improvement in patients without active bleeding during the use of terlipressin. RESULTS: During the use of terlipressin, CysC value was significantly reduced (1.3±0.8 vs. 1.1±0.7, P=0.001); Cr value was reduced, but the reduction was not statistically significant (68.8±24 vs. 65.5±23, P=0.817); the rate of CysC reduction was significantly higher in patients treated with terlipressin than those treated with somatostatin/octreotide (73.1% vs. 0%, P=0.005); the rate of Cr reduction was not significantly different between patients treated with terlipressin and somatostatin/octreotide (61.5% vs. 20%, P=0.148); no factor associated with CysC reduction was identified; higher hemoglobin, red blood cell, and platelet and lower prothrombin time and international normalized ratio at baseline were significantly associated with Cr reduction. After terlipressin was stopped, neither CysC nor Cr value was significantly reduced (P=0.852 and P=0.296). CONCLUSIONS: Terlipressin may be beneficial on preventing renal function impairment in cirrhotic patients with AUGIB.
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METHODS: Cirrhotic patients who were consecutively hospitalized between January 2016 and March 2019 were screened. Serum cardiac biomarkers at admission, including N-Terminal pro-B-type natriuretic peptide (NT-pro BNP), high-sensitivity cardiac troponin T (hs-cTnT), creatine kinase (CK), creatine kinase MB (CK-MB), and lactate dehydrogenase (LDH), were collected. Acute decompensating events at admission, primarily including ascites, acute gastrointestinal hemorrhage, and acute-on-chronic liver failure (ACLF), were recorded. RESULTS: The NT-pro BNP level was significantly higher in cirrhotic patients with acute decompensating events than in those without any decompensating events (median: 140.75 pg/mL versus 41.86 pg/mL, P < 0.001). The NT-pro BNP level significantly correlated with ascites, acute gastrointestinal hemorrhage, and ACLF. The hs-cTnT level was significantly higher in cirrhotic patients with acute decompensating events than in those without decompensating events (median: 0.008 ng/mL versus 0.006 ng/mL, P = 0.007). The hs-cTnT level significantly correlated with acute gastrointestinal hemorrhage, but not ascites or ACLF. LDH (185.0 U/L versus 173.5 U/L, P = 0.281), CK (71 U/L versus 84 U/L, P = 0.157), and CK-MB (29.5 U/L versus 33.0 U/L, P = 0.604) levels were not significantly different between cirrhotic patients with and without acute decompensating events. CONCLUSION: The elevated NT-pro BNP level seems to be closely related to the development of acute decompensating events in liver cirrhosis.
RESUMO
Hepatorenal syndrome (HRS) is a serious complication of liver cirrhosis, which is of pre-renal origin due to central volume depletion together with cardiac dysfunction and characterized by oliguria with severe urinary sodium retention and elevated serum creatinine levels. HRS is divided into HRS I, which is rapidly progressive and mostly seen in patients with decompensated liver cirrhosis, and HRS II, which progresses more slowly and is always accompanied by gross ascites. Liver transplantation is the best choice of treatment for HRS but rarely available. Current mainstay pharmacological therapies are vasoconstrictors, such as terlipressin, noradrenaline and dopamine, in combination with albumin. This paper aims to overview the current evidence regarding outcomes of terlipressin for the treatment of HRS.