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Hyperexcitability is associated with neuronal dysfunction, cellular death, and consequently neurodegeneration. Redox disbalance can contribute to hyperexcitation and increased reactive oxygen species (ROS) levels are observed in various neurological diseases. NOX4 is an NADPH oxidase known to produce ROS and might have a regulating function during oxidative stress. We, therefore, aimed to determine the role of NOX4 on neuronal firing, hyperexcitability, and hyperexcitability-induced changes in neural network function. Using a multidimensional approach of an in vivo model of hyperexcitability, proteomic analysis, and cellular function analysis of ROS, mitochondrial integrity, and calcium levels, we demonstrate that NOX4 is neuroprotective by regulating ROS and calcium homeostasis and thereby preventing hyperexcitability and consequently neuronal death. These results implicate NOX4 as a potential redox regulator that is beneficial in hyperexcitability and thereby might have an important role in neurodegeneration.
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Cálcio , Proteômica , Humanos , NADPH Oxidase 4 , NADPH Oxidases/metabolismo , Estresse Oxidativo , Espécies Reativas de OxigênioRESUMO
OBJECTIVE: To analyze the concerns and worries about planning to have children and being a parent as a person with epilepsy and investigate gender differences in these perceptions. METHODS: The Epi2020 study was a large multicenter study focusing on different healthcare aspects of adult patients with epilepsy in Germany. In addition to basic clinical and demographic characteristics, patients were asked to answer a questionnaire regarding their plan to have children, if they had children, and concerns about their children's health. Data were analyzed to detect differences between men and women with epilepsy according to age group. RESULTS: In total, 477 patients with epilepsy with a mean age of 40.5â¯years (SDâ¯=â¯15.5, range: 18-83â¯years) participated in this study; 280 (58.7%) were female and 197 (41.3%) were male. Both women and men frequently reported concerns and worries about having children: In the age group below 45â¯years of age, 72.5% of women and 58.2% of men described being worried to some extent that their children may also suffer from epilepsy (pâ¯=â¯.006). Furthermore, 67.3% of women and 54.2% of men below the age of 45â¯years reported being worried that their children may be disabled (pâ¯=â¯.003). Women were more likely to have family members who are reluctant to support their desire to have children (pâ¯=â¯.048). CONCLUSION: Women with epilepsy of childbearing age are significantly more likely to report major concerns that their children might be disabled or also have epilepsy than men with epilepsy and, therefore, express more concerns about choosing to have a child. However, men also report frequent concerns and worries, and this should be addressed not only on request but should be included in the provision of general information on epilepsy.
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Epilepsia , Adulto , Idoso , Epilepsia/epidemiologia , Feminino , Alemanha/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Pais , Estudos Prospectivos , Fatores SexuaisRESUMO
Endothelial cell (EC) metabolism is emerging as a regulator of angiogenesis, but the precise role of glutamine metabolism in ECs is unknown. Here, we show that depriving ECs of glutamine or inhibiting glutaminase 1 (GLS1) caused vessel sprouting defects due to impaired proliferation and migration, and reduced pathological ocular angiogenesis. Inhibition of glutamine metabolism in ECs did not cause energy distress, but impaired tricarboxylic acid (TCA) cycle anaplerosis, macromolecule production, and redox homeostasis. Only the combination of TCA cycle replenishment plus asparagine supplementation restored the metabolic aberrations and proliferation defect caused by glutamine deprivation. Mechanistically, glutamine provided nitrogen for asparagine synthesis to sustain cellular homeostasis. While ECs can take up asparagine, silencing asparagine synthetase (ASNS, which converts glutamine-derived nitrogen and aspartate to asparagine) impaired EC sprouting even in the presence of glutamine and asparagine. Asparagine further proved crucial in glutamine-deprived ECs to restore protein synthesis, suppress ER stress, and reactivate mTOR signaling. These findings reveal a novel link between endothelial glutamine and asparagine metabolism in vessel sprouting.
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Asparagina/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Glutamina/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Meios de Cultura/química , Células Endoteliais/metabolismo , Glutaminase/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Redes e Vias Metabólicas , Neovascularização PatológicaRESUMO
The importance of endothelial cell (EC) metabolism and its regulatory role in the angiogenic behavior of ECs during vessel formation and in the function of different EC subtypes determined by different vascular beds has been recognized only in the last few years. Even more importantly, apart from a role of nitric oxide and reactive oxygen species in EC dysfunction, deregulations of EC metabolism in disease only recently received increasing attention. Although comprehensive metabolic characterization of ECs still needs further investigation, the concept of targeting EC metabolism to treat vascular disease is emerging. In this overview, we summarize EC-specific metabolic pathways, describe the current knowledge on their deregulation in vascular diseases, and give an outlook on how vascular endothelial metabolism can serve as a target to normalize deregulated endothelium.
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Células Endoteliais/metabolismo , Doenças Vasculares/metabolismo , Animais , Arginina/metabolismo , Ácidos Graxos/metabolismo , Glutamina/metabolismo , Glicogênio/metabolismo , Glicólise , Hexosaminas/biossíntese , Humanos , Neovascularização Patológica , Neovascularização Fisiológica , Via de Pentose Fosfato , Doenças Vasculares/complicaçõesRESUMO
Angiogenesis and inflammation go hand in hand in various (patho-)physiological conditions. Several studies have highlighted the interconnection between endothelial cells (ECs) and macrophages in these conditions at the level of growth factor and cytokine signaling, yet the importance of metabolism and metabolic signaling has been largely overlooked. Modulating macrophage and/or endothelial functions by interfering with metabolic pathways offers new perspectives for therapeutic strategies. In this review, we highlight the complexity of the interrelationship between the inflammatory response and angiogenesis. More in particular, the interaction between macrophages and ECs will be discussed with a special focus on how their metabolism can contribute to (patho-)physiological conditions.
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Células Endoteliais/fisiologia , Macrófagos/fisiologia , Transdução de Sinais/fisiologia , Animais , Humanos , Inflamação/fisiopatologia , Neovascularização Patológica/fisiopatologia , Neovascularização Fisiológica/fisiologiaRESUMO
BACKGROUND: Procalcitonin is used as a diagnostic tool for the identification and risk stratification of septic patients. Procalcitonin plasma concentrations tightly correlate with the severity of the ongoing inflammatory reaction and can rise up to 10,000-fold. Impairment of endothelial cell function plays an important role in the pathogenesis of hypotension and disturbed organ perfusion during sepsis. We investigated the possible effects of procalcitonin itself on endothelial cell function and viability. METHODS: Human endothelial cells were exposed to 0.01 to 100 ng/mL procalcitonin and investigated for endothelial permeability using transwells, migration in a scratch wound assay and new capillary formation on extracellular matrix in vitro. Tumor necrosis factor-α and vascular endothelial growth factor served as positive controls. Procalcitonin's impact on the response of endothelial cells toward ischemia was investigated in vivo in the murine model of unilateral femoral artery ligation. Procalcitonin-exposed endothelial cells were subjected to immunoblot for the investigation of vascular endothelial-cadherin expression and angiogenic signaling pathways. Flow cytometry was used for the detection of inflammatory activation and viability, and genomic analysis was performed. Data are presented as difference in means and 95% confidence intervals; statistical analyses were performed using analysis of variance/Bonferroni, and P values are reported as adjusted for multiple comparisons (Padjust). RESULTS: Tumor necrosis factor-α and 0.1 ng/mL procalcitonin induced endothelial barrier disruption after incubation of endothelial monolayers for 6 hours (-2.53 [-4.16 to -0.89], P = .0008 and -2.09 [-3.73 to -0.45], Padjust = .0064 compared with vehicle-treated control, respectively). Procalcitonin beginning at concentrations of 0.02 ng/mL reduced endothelial cell migration (0.26 [0.06 to 0.47], Padjust = .0069) and new capillary formation in vitro (0.47 [0.28 to 0.66], Padjust < .0001) contrasting the proangiogenic action of vascular endothelial growth factor. Left ventricular injection of procalcitonin in mice on postoperative day 1, 3, and 5 after induction of ischemia impaired new capillary formation and recovery of hindlimb perfusion in vivo (number of capillaries/mm in the ischemic leg of vehicle-treated versus procalcitonin-treated mice, 852.6 [383.4-1322], Padjust = .0002). Twenty-four-hour incubation with procalcitonin reduced the expression of vascular endothelial-cadherin at 100 ng/mL (0.39 [0.06-0.71], Padjust = .0167) and induced endothelial cell death (apoptosis, -5.4 [-10.67 to -0.13], Padjust = .0431). No alteration in the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1 or extracellular signal-regulated kinase 1/2, and AKT signaling pathways was observed. Genomic analysis revealed regulation of a variety of genes involved in inflammation, angiogenesis, and cell growth. CONCLUSIONS: This study found that procalcitonin itself impaired several aspects of endothelial cell function. Procalcitonin-induced loss of endothelial barrier function may contribute to capillary leakage and therapy-refractory hypotension during sepsis. Anti-angiogenic properties of procalcitonin at low concentrations could also identify procalcitonin as a mediator of vascular disease associated with the metabolic syndrome. Future studies are needed to further test procalcitonin as a potential therapeutic target for preserving vascular dysfunction during acute and chronic inflammatory disorders.
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Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Calcitonina/toxicidade , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Isquemia/induzido quimicamente , Isquemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
OBJECTIVE: Toll-like receptor 2 (TLR2) inhibition by function blocking antibodies (ABs) is associated with enhanced preservation of endothelial cell function during vascular disease. In the present study, we investigated the capacity of TLR2-blocking ABs to modulate the angiogenic response of endothelial cells in vitro and in vivo. APPROACH AND RESULTS: Incubation of endothelial cells with mono- or polyclonal anti-TLR2 ABs resulted in increased tube formation, sprouting, and migration of endothelial cells compared with controls. In a mouse model of hindlimb ischemia, using TLR2-deficient or anti-TLR2 AB-treated wild-type mice resulted in increased new capillary formation and enhanced reperfusion. The effects of anti-TLR2 ABs were similar to those exerted by stromal cell-derived factor-1, and we show that anti-TLR2 ABs yet not TLR2 ligands lead to comparable activation of extracellular signal-regulated kinase1/2 and AKT but not p38 mitogen-activated protein kinase as activation of the CXCR4 canonical signal transduction pathways by stromal cell-derived factor-1. Immunoprecipitation of TLR2 revealed that anti-TLR2 ABs initiate an association of TLR2 with CXCR4 and mitogen-activated protein kinase activation. The proangiogenic properties of anti-TLR2 ABs were abolished by both G-protein inhibition and CXCR4 knockdown in endothelial cells. CONCLUSIONS: Our results provide evidence for a proangiogenic effect of TLR2-blocking ABs on endothelial cells in vitro and in vivo. They identify a novel molecular mechanism linking TLR2 to angiogenic processes that is independent from the activation of inflammatory cascades and further support the concept of a beneficial effect of TLR2 inhibition for endothelial cell function in vascular disease.
Assuntos
Anticorpos Bloqueadores/farmacologia , Sistema de Sinalização das MAP Quinases/imunologia , Neovascularização Fisiológica/imunologia , Doença Arterial Periférica/imunologia , Receptores CXCR4/metabolismo , Receptor 2 Toll-Like/imunologia , Animais , Células Cultivadas , Quimiocina CXCL12/metabolismo , Células Endoteliais/citologia , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Membro Posterior/irrigação sanguínea , Isquemia/imunologia , Isquemia/metabolismo , Isquemia/fisiopatologia , Camundongos , Camundongos Knockout , Músculo Esquelético/irrigação sanguínea , Doença Arterial Periférica/metabolismo , Doença Arterial Periférica/fisiopatologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Receptores CXCR4/genética , Receptores CXCR4/imunologia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Epilepsy is one of the most common neurological disorders in the world. Common epileptic drugs generally affect ion channels or neurotransmitters and prevent the emergence of seizures. However, up to a third of the patients suffer from drug-resistant epilepsy, and there is an urgent need to develop new therapeutic strategies that go beyond acute antiepileptic (antiseizure) therapies towards therapeutics that also might have effects on chronic epilepsy comorbidities such as cognitive decline and depression. The mitochondrial calcium uniporter (MCU) mediates rapid mitochondrial Ca2+ transport through the inner mitochondrial membrane. Ca2+ influx is essential for mitochondrial functions, but longer elevations of intracellular Ca2+ levels are closely associated with seizure-induced neuronal damage, which are underlying mechanisms of cognitive decline and depression. Using neuronal-specific MCU knockout mice (MCU-/-ΔN), we demonstrate that neuronal MCU deficiency reduced hippocampal excitability in vivo. Furthermore, in vitro analyses of hippocampal glioneuronal cells reveal no change in total Ca2+ levels but differences in intracellular Ca2+ handling. MCU-/-ΔN reduces ROS production, declines metabolic fluxes, and consequently prevents glioneuronal cell death. This effect was also observed under pathological conditions, such as the low magnesium culture model of seizure-like activity or excitotoxic glutamate stimulation, whereby MCU-/-ΔN reduces ROS levels and suppresses Ca2+ overload seen in WT cells. This study highlights the importance of MCU at the interface of Ca2+ handling and metabolism as a mediator of stress-related mitochondrial dysfunction, which indicates the modulation of MCU as a potential target for future antiepileptogenic therapy.
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BACKGROUND: Seizure clusters, prolonged seizures, and status epilepticus are life-threatening neurological emergencies leading to irreversible neuronal damage. Benzodiazepines are current evidence-based rescue therapy options; however, recent investigations indicated the prescription of mainly unsuitable benzodiazepines and inappropriate use of rescue medication. OBJECTIVE: To examine current use, satisfaction, and adverse events concerning rescue medication in patients with epilepsy in Germany. PATIENTS AND METHODS: The study was conducted at epilepsy centres in Frankfurt am Main, Greifswald, Marburg, and Münster between 10/2020 and 12/2020. Patients with an epilepsy diagnosis were assessed based on a questionnaire examining a 12-month period. RESULTS: In total, 486 patients (mean age: 40.5, range 18-83, 58.2 % female) participated in this study, of which 125 (25.7 %) reported the use of rescue medication. The most frequently prescribed rescue medications were lorazepam tablets (56.8 %, n = 71 out of 125), buccal midazolam (19.2 %, n = 24), and rectal diazepam (10.4 %, n = 13). Seizures continuing for over several minutes (43.2 %, n = 54), seizure clusters (28.0 %, n = 35), and epileptic auras (28.0 %, n = 35) were named as indications, while 28.0 % (n = 35) stated they administered the rescue medication for every seizure. Of those continuing to have seizures, 46.0 % did not receive rescue medication. On average, rescue medication prescription occurred 7.1 years (SD 12.7, range 0-66) after an epilepsy diagnosis. CONCLUSIONS: Unsuitable oral benzodiazepines remain widely prescribed for epilepsy patients as rescue medication. Patients also reported inappropriate use of medication. A substantial proportion of patients who were not seizure-free did not receive rescue medication prescriptions. Offering each patient at risk for prolonged seizures or clusters of seizures an individual rescue treatment with instructions on using it may decrease mortality and morbidity and increase quality of life. .
Assuntos
Anticonvulsivantes , Epilepsia , Humanos , Adulto , Feminino , Masculino , Alemanha , Estudos Transversais , Pessoa de Meia-Idade , Anticonvulsivantes/uso terapêutico , Anticonvulsivantes/administração & dosagem , Idoso , Adulto Jovem , Adolescente , Epilepsia/tratamento farmacológico , Idoso de 80 Anos ou mais , Benzodiazepinas/uso terapêutico , Lorazepam/uso terapêutico , Midazolam/uso terapêutico , Midazolam/administração & dosagemRESUMO
Hyperexcitability-induced neuronal damage plays a role both in epilepsy as well as in inflammatory brain diseases such as multiple sclerosis (MS) and as such represents an important disease pathway which potentially can be targeted to mitigate neuronal damage. Dimethyl fumarate (DMF) and its pharmacologically active metabolite monomethyl fumarate (MMF) are FDA-approved therapeutics for MS, which can induce immunosuppressive and antioxidant pathways, and their neuroprotective capacity has been demonstrated in other preclinical neurological disease models before. In this study, we used an unbiased proteomic approach to identify potential new targets upon the treatment of MMF in glio-neuronal hippocampal cultures. MMF treatment results in induction of antioxidative (HMOX1, NQO1) and anaplerotic metabolic (GAPDH, PC) pathways, which correlated with reduction in ROS production, increased mitochondrial NADH-redox index and decreased NADH pool, independent of glutathione levels. Additionally, MMF reduced glycolytic capacity indicating individual intra-cellular metabolic programs within different cell types. Furthermore, we demonstrate a neuroprotective effect of MMF upon hyperexcitability in vitro (low magnesium model), where MMF prevents glio-neuronal death via reduced ROS production. These results highlight MMF as a potential new therapeutic opportunity in hyperexcitability-induced neurodegeneration.
Assuntos
Antioxidantes , Fármacos Neuroprotetores , Antioxidantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo , NAD , Proteômica , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Fator 2 Relacionado a NF-E2/metabolismoRESUMO
BACKGROUND: Assessment of quality of life (QoL) has become an important indicator for chronic neurological diseases. While these conditions often limit personal independence and autonomy, they are also associated with treatment-related problems and reduced life expectancy. Epilepsy has a tremendous impact on the QoL of patients and their families, which is often underestimated by practitioners. The aim of this work was to identify relevant factors affecting QoL in adults with epilepsy. METHODS: This cross-sectional, multicenter study was conducted at four specialized epilepsy centers in Germany. Patients diagnosed with epilepsy completed a standardized questionnaire focusing on QoL and aspects of healthcare in epilepsy. Univariate regression analyses and pairwise comparisons were performed to identify variables of decreased QoL represented by the overall Quality of Life in Epilepsy Inventory (QOLIE-31) score. The variables were then considered in a multivariate regression analysis after multicollinearity analysis. RESULTS: Complete datasets for the QOLIE-31 were available for 476 patients (279 [58.6%] female, 197 [41.4%] male, mean age 40.3 years [range 18-83 years]). Multivariate regression analysis revealed significant associations between low QoL and a high score on the Liverpool Adverse Events Profile (LAEP; beta=-0.28, p < 0.001), Hospital Anxiety and Depression Scale - depression subscale (HADS-D; beta=-0.27, p < 0.001), Neurological Disorders Depression Inventory in Epilepsy (NDDI-E; beta=-0.19, p < 0.001), revised Epilepsy Stigma Scale (beta=-0.09, p = 0.027), or Seizure Worry Scale (beta=-0.18, p < 0.001) and high seizure frequency (beta = 0.14, p < 0.001). CONCLUSION: Epilepsy patients had reduced QoL, with a variety of associated factors. In addition to disease severity, as measured by seizure frequency, the patient's tolerability of anti-seizure medications and the presence of depression, stigma, and worry about new seizures were strongly associated with poor QoL. Diagnosed comorbid depression was underrepresented in the cohort; therefore, therapeutic decisions should always consider individual psychobehavioral and disease-specific aspects. Signs of drug-related adverse events, depression, fear, or stigmatization should be actively sought to ensure that patients receive personalized and optimized treatment. TRIAL REGISTRATION: German Clinical Trials Register (DRKS00022024; Universal Trial Number: U1111-1252-5331).
RESUMO
Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system that causes demyelination, axonal degeneration and astrogliosis, resulting in progressive neurological disability. Fuelled by an evolving understanding of MS immunopathogenesis, the range of available immunotherapies for clinical use has expanded over the past two decades. However, MS remains an incurable disease and even targeted immunotherapies often fail to control insidious disease progression, indicating the need for new and exceptional therapeutic options beyond the established immunological landscape. In this Review, we highlight such non-canonical targets in preclinical MS research with a focus on five highly promising areas: oligodendrocytes; the blood-brain barrier; metabolites and cellular metabolism; the coagulation system; and tolerance induction. Recent findings in these areas may guide the field towards novel targets for future therapeutic approaches in MS.
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Esclerose Múltipla , Progressão da Doença , Humanos , Esclerose Múltipla/tratamento farmacológicoRESUMO
OBJECTIVE: Epilepsy is a chronic condition that can affect patients of all ages. Women with epilepsy (WWE) require access to specific counseling and information regarding issues related to contraception, pregnancy, and hormonal effects on seizure control and bone mineral density. This study investigated the knowledge among WWE regarding their condition, and whether epilepsy-specific knowledge has improved over the last 15 years. METHODS: A total of 280 WWE aged 18 to 82 years participated in this multicenter, questionnaire-based study. The study was conducted at four epilepsy centers in Germany, between October 2020 and December 2020. Sociodemographic and epilepsy-specific data for participating women were analyzed and compared with the results of a similar survey performed in 2003-2005 among 365 WWE in Germany. RESULTS: The questionnaire-based survey revealed considerable knowledge deficits without significant improvements over the last 15 years, particularly among those with less education and with regards to information on the more pronounced effects of epilepsy in older WWE (>50 years), including interactions with menopause and osteoporosis. In WWE ≤29 years, a significant increase in the knowledge score was observed in 2020 compared with this age group in 2005 (mean 7.42 vs. 6.5, p = .036). Mothers frequently reported epilepsy-related concerns regarding childrearing, particularly of seizures scaring their child and the need to rely on other people. CONCLUSION: WWE continue to demonstrate inadequate epilepsy-related knowledge. Despite increasing information availability and the aspiration toward better awareness among medical professionals, overall knowledge has not increased sufficiently compared with the levels observed in recent studies.
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Epilepsia , Complicações na Gravidez , Idoso , Anticonvulsivantes/uso terapêutico , Anticoncepção , Epilepsia/tratamento farmacológico , Feminino , Alemanha/epidemiologia , Humanos , Gravidez , Complicações na Gravidez/tratamento farmacológico , Convulsões/tratamento farmacológicoRESUMO
BACKGROUND: Multiple sclerosis is one of the most prevalent neurological diseases in young adults affecting over 2 million people worldwide. Alemtuzumab is a highly effective therapy in relapsing remitting MS. Alemtuzumab is a monoclonal CD52 antibody that proved its efficacy against an active comparator (interferon [IFN]-ß1a) in a phase II trial and two phase III trials regarding clinical and MRI outcomes. Nevertheless, the exact mode of action is still unknown. Alemtuzumab is commonly associated with secondary autoimmune disorders significantly affecting the risk-benefit ratio. Therefore, new biomarkers predicting treatment response and adverse events are urgently needed. This study aims to further elucidate the mechanism of action of the neuroprotective potential of alemtuzumab in relapsing-remitting multiple sclerosis (RRMS). METHODS/DESIGN: This is a 3-year multicentre, explorative study including overall 150 patients comprising three different groups: (i) de novo patients prior and after alemtuzumab treatment initiation, (ii) patients under alemtuzumab treatment and (iii) patients requiring more than two alemtuzumab infusions. Peripheral blood and serum samples will be collected semi-annually for several in vitro/ex vivo assays to detect and characterize immune cells including their functional activity. Furthermore, data of MRI scans and disease-related impairment (using EDSS and MSFC), as well as the number and time of relapses, will be assessed. The clinical study is registered at clinicaltrials.gov (NCT04082260). PERSPECTIVE: Our study will provide deep insights into the underlying immunological changes in a longitudinal analysis of alemtuzumab treated RRMS patients. By combining clinical, radiological and functional immune-phenotype data, we will be able to identify biomarkers and/or immune signatures predicting treatment response and adverse events. Thereby, the understanding of the mechanisms of action of alemtuzumab will improve its efficacy and safety for present and future patients.
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Little is known about the metabolism of quiescent endothelial cells (QECs). Nonetheless, when dysfunctional, QECs contribute to multiple diseases. Previously, we demonstrated that proliferating endothelial cells (PECs) use fatty acid ß-oxidation (FAO) for de novo dNTP synthesis. We report now that QECs are not hypometabolic, but upregulate FAO >3-fold higher than PECs, not to support biomass or energy production but to sustain the tricarboxylic acid cycle for redox homeostasis through NADPH regeneration. Hence, endothelial loss of FAO-controlling CPT1A in CPT1AΔEC mice promotes EC dysfunction (leukocyte infiltration, barrier disruption) by increasing endothelial oxidative stress, rendering CPT1AΔEC mice more susceptible to LPS and inflammatory bowel disease. Mechanistically, Notch1 orchestrates the use of FAO for redox balance in QECs. Supplementation of acetate (metabolized to acetyl-coenzyme A) restores endothelial quiescence and counters oxidative stress-mediated EC dysfunction in CPT1AΔEC mice, offering therapeutic opportunities. Thus, QECs use FAO for vasculoprotection against oxidative stress-prone exposure.
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Carnitina O-Palmitoiltransferase/metabolismo , Metabolismo Energético , Ácidos Graxos/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , NADP/metabolismo , Receptor Notch1/metabolismo , Animais , Proliferação de Células , Células HEK293 , Homeostase , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , Estresse OxidativoRESUMO
BACKGROUND: Protein Z (PZ) is a vitamin K-dependent coagulation factor without catalytic activity. Evidence points towards PZ as an independent risk factor for the occurrence of human atherosclerotic vascular diseases. The aim of this study was to investigate the role of PZ in vascular arterial disease. MATERIAL AND METHODS: PZ-deficient (PZ(-/-)) mice and their wild-type littermates (PZ(+/+)) were subjected to unilateral carotid artery injury by using ferric chloride and dissected 21 days thereafter for histological analysis. Human aortic smooth muscle cells (SMC) were used for in vitro wound healing assay to assess the influence of PZ on SMC migration and for cell proliferation studies. RESULTS: Morphometric analysis of neointima formation revealed a significantly increased area and thickness of the neointima and subsequently increased luminal stenosis in carotid arteries of PZ(-/-) mice compared to PZ(+/+) mice (p < 0.05, n = 9). Immunohistochemical analysis of neointima lesion composition revealed significantly higher numbers of PCNA-positive and α-SMA-positive cells in the neointima of PZ(-/-) mice. Furthermore, PZ showed an anti-migratory potency in in vitro wound healing assay with SMCs, while no effect of PZ on SMC proliferation was detectable. Conclusion: PZ contributes to a reduced neointima formation after vascular injury, underlining the modulatory role of the coagulation cascade in vascular homeostasis.
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Proteínas Sanguíneas/deficiência , Lesões das Artérias Carótidas/metabolismo , Inflamação/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neointima , Lesões do Sistema Vascular/metabolismo , Actinas/metabolismo , Animais , Proteínas Sanguíneas/genética , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/induzido quimicamente , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Cloretos , Modelos Animais de Doenças , Compostos Férricos , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/lesões , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fatores de Tempo , Lesões do Sistema Vascular/induzido quimicamente , Lesões do Sistema Vascular/genética , Lesões do Sistema Vascular/patologiaRESUMO
OBJECTIVE: Toll-like receptor 2 (TLR2)-deficiency is associated with the preservation of vascular function and TLR2-deficient (TLR2(-/-)) mice exhibit increased neovascularization following induction of hindlimb ischemia. Hematopoietic stem cells play an important role in ischemia-induced angiogenesis and we now investigated whether the effects observed in TLR2(-/-) mice may be attributed to TLR2 deficiency on bone marrow-derived stem cells. APPROACH AND RESULTS: cKit-positive (cKit(+)) bone marrow cells (BMC) were isolated from wild type (WT) and TLR2(-/-) mice employing MACS-bead technology. Co-incubation of TLR2(-/-)cKit(+) BMC with mature endothelial cells (ECs) resulted in increased tube formation of ECs on matrigel, augmented sprouting in a 3D-collagen matrix and increased migratory capacity compared to co-incubation with WT cKit(+) BMC. In an in vivo matrigel plug assay, TLR2(-/-)cKit(+) BMC exhibited enhanced formation of capillary-like networks. In a murine model of hindlimb ischemia, administration of TLR2(-/-) cKit(+) BMC to WT mice augmented capillary density and reperfusion of ischemic M. gastrocnemius muscle tissue to the level of TLR2(-/-) mice. Western Blot analysis revealed comparable expression of CXCR4 on TLR2(-/-)cKit(+) BMC but increased activation of the PI3K downstream signaling molecule protein kinase B (PKB/AKT) compared to WT cKit(+) cells. CONCLUSIONS: The absence of TLR2 on cKit(+) BMC is associated with augmented potency to support angiogenic processes in vitro and in vivo. Functional inhibition of TLR2 may therefore provide a novel tool to enhance stem cell function for the treatment of vascular diseases.