RESUMO
Periods of low energy supply are challenging conditions for organisms and cells during fasting or famine. Although changes in nutrient levels in the blood are first sensed by endothelial cells, studies on their metabolic adaptations to diminished energy supply are lacking. We analyzed the dynamic metabolic activity of human umbilical vein endothelial cells (HUVECs) in basal conditions and after serum starvation. Metabolites of glycolysis, the tricarboxylic acid (TCA) cycle, and the glycerol pathway showed lower levels after serum starvation, whereas amino acids had increased levels. A metabolic flux analysis with 13C-glucose or 13C-glutamine labeling for different time points reached a plateau phase of incorporation after 30 h for 13C-glucose and after 8 h for 13C-glutamine under both experimental conditions. Notably, we observed a faster label incorporation for both 13C-glucose and 13C-glutamine after serum starvation. In the linear range of label incorporation after 3 h, we found a significantly faster incorporation of central carbon metabolites after serum starvation compared to the basal state. These findings may indicate that endothelial cells develop increased metabolic activity to cope with energy deficiency. Physiologically, it can be a prerequisite for endothelial cells to form new blood vessels under unfavorable conditions during the process of angiogenesis in vivo.
Assuntos
Glutamina , Inanição , Humanos , Glutamina/metabolismo , Aminoácidos/metabolismo , Glicólise , Glucose/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismoRESUMO
There is growing evidence for sex and gender differences in the clinical manifestation and outcomes of human diseases. Human primary endothelial cells represent a useful cardiovascular model to study sexual dimorphisms at the cellular level. Here, we analyzed sexual dimorphisms of the secretome after serum starvation using human umbilical vein endothelial cells (HUVECs) from twin pairs of the opposite sex to minimize the impact of varying genetic background. HUVECs were starved for 5 and 16 h, respectively, and proteins of the cell culture supernatants were analyzed by tandem mass spectrometry. Altogether, 960 extracellular proteins were identified of which 683 were amendable to stringent quantification. Significant alterations were observed for 455 proteins between long-term and short-term starvation and the majority were similar in both sexes. Only 5 proteins showed significant sex-specific regulation between long-versus short-term starvation. Furthermore, 19 unique proteins with significant sexual dimorphisms at the same time points of serum starvation were observed. A larger number of proteins, for example tissue factor inhibitor 2 (TFPI2), displayed higher levels in the supernatants of females compared to male cells after long term serum starvation that might point to higher adaptation capacity of female cells. The overall results demonstrate that male and female cells differ in their secretome.
Assuntos
Proteínas , Caracteres Sexuais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Proteínas/metabolismo , Fatores SexuaisRESUMO
VEGF inhibition in gastric cancer has a proven benefit in the second line setting. Pazopanib, an oral tyrosine kinase inhibitor, selectively inhibits VEGFR-1, -2 and -3, c-kit and PDGF-R resulting in inhibition of angiogenesis. This open-label randomized phase II trial (2:1) investigated the efficacy of combining pazopanib with FLO (5-fluorouracil, oxaliplatin) vs FLO alone (internal control arm) as first-line treatment in patients with advanced adenocarcinoma of the stomach and gastroesophageal junction (GEJ). Eighty-seven patients were randomized and 78 patients were eligible and evaluable (PaFLO arm 51 patients, FLO arm 27 patients). The PFS rate at 6 months (primary endpoint) was 34% in the PaFLO arm vs 30% in the FLO arm. Comparing PaFLO with FLO median PFS was 4.66 months (95% confidence interval [CI] 2.87-6.46) vs 4.47 months (95% CI 1.79-7.14) (95% CI, hazard ratio [HR] 0.96 (0.60-1.55), P = .882 [exploratory]); median OS was 10.19 months (95% CI 5.46-14.92) vs 7.33 months (95% CI 4.93-9.73), (95% CI HR 1.01 [0.62-1.65], P = .953, exploratory), disease control rate was 72% vs 59%. PaFLO was well tolerable, toxicities were slightly higher in the PaFLO arm. Major adverse events were loss of appetite, nausea, fatigue, diarrhea, neutropenia and thrombocytopenia. Adding pazopanib to chemotherapy shows signs of efficacy but no major improvement in this randomized phase 2 trial. The PFS at 6 months in both arms was lower than expected from the literature. Biomarkers identifying subgroups who benefit and novel combinations are needed. ClinicalTrials.gov: NCT01503372.
Assuntos
Adenocarcinoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Gástricas/tratamento farmacológico , Idoso , Junção Esofagogástrica/patologia , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/efeitos adversos , Humanos , Indazóis/administração & dosagem , Indazóis/efeitos adversos , Masculino , Pessoa de Meia-Idade , Oxaliplatina/administração & dosagem , Oxaliplatina/efeitos adversos , Pirimidinas/administração & dosagem , Pirimidinas/efeitos adversos , Neoplasias Gástricas/mortalidade , Sulfonamidas/administração & dosagem , Sulfonamidas/efeitos adversosRESUMO
The underlying mechanisms for the vasodilating effects of the tea catechin epigallocatechin-3-gallate (EGCG) are still not fully understood. Besides nitric oxide (NO)-dependent effects, other modes of action are discussed. To elucidate whether the NO pathway is a prerequisite in mediating vasodilating effects, we investigated EGCG-induced vasorelaxation in isolated aortic rings of endothelial nitric oxide knockout (eNOS) mice. Vasodilation to acetylcholine was fully prevented in aortic rings of eNOS mice, confirming lack of vascular NO production. Vasodilation to the exogenous NO donor sodium nitroprusside was preserved in eNOS mice aortic rings. Low concentrations of EGCG (5-15 µM) resulted in strong vasorelaxation in aortic rings of wild type mice, whereas it was completely absent in eNOS mice. In corroboration, relaxation in response to green tea was significantly inhibited in aortic rings of eNOS mice. These results demonstrate that EGCG-induced vasodilation strongly relies on functional NO synthase in endothelial cells and subsequent stimulation of NO production in vessels.
Assuntos
Aorta/efeitos dos fármacos , Catequina/análogos & derivados , Óxido Nítrico Sintase Tipo III/deficiência , Óxido Nítrico/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Aorta/enzimologia , Catequina/farmacologia , Relação Dose-Resposta a Droga , Técnicas In Vitro , Masculino , Camundongos Knockout , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo III/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Eicosanoids and docosanoids have been shown to be involved in atherosclerosis. Polyunsaturated fatty acids (PUFAs) are important nutrients that are metabolized by lipoxygenases and cyclooxygenases to various mono-hydroxy metabolites which can be further metabolized by specific enzymes to more complex eicosanoids and docosanoids. In this study a high-performance liquid chromatography methodology was established and rabbits were fed with a control or a high-cholesterol diet to induce atherosclerotic lesions to determine pro- or anti-inflammatory lipid mediators in atherosclerotic vessels. In aortic samples from atherosclerotic rabbits we determined for the first time various eicosanoids/docosanoids and observed an increased concentration of 12-lipoxygenase metabolites. Increased levels of 12-hydroxyeicosatetraenoic acid (12-HETE) in high-cholesterol versus control animals as well as increased ratios of 12-HETE/arachidonic acid ratios indicate that 12-lipoxygenase metabolites may have importance in atherosclerosis. In addition, decreased concentrations of the 5-lipoxygenase metabolite leukotriene B4 levels were detected in high-cholesterol animals. A positive correlation of total plaque area with plasma levels of 12-HETE and a negative correlation with aortic levels of endogenous PPARγ-ligand 13-oxo-octadecadienoic acid were found. This study let us conclude that the cholesterol content in the diet might influence atherosclerosis via increased 12-lipoxygenase- and cyclooxygenase-mediated pathways and reduced 5-lipoxygenase pathways.
Assuntos
Aorta/metabolismo , Aterosclerose/fisiopatologia , Colesterol na Dieta/farmacologia , Ácidos Docosa-Hexaenoicos/metabolismo , Eicosanoides/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Animais , Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 5-Lipoxigenase/metabolismo , Aterosclerose/sangue , Aterosclerose/patologia , Cromatografia Líquida de Alta Pressão/métodos , Ácidos Docosa-Hexaenoicos/sangue , Eicosanoides/sangue , Ácidos Graxos Insaturados/sangue , Masculino , Redes e Vias Metabólicas , Placa Aterosclerótica/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Coelhos , Espectrometria de Massas em Tandem/métodosRESUMO
Virtual Reality (VR) is applied in various areas were a high User Experience is essential. The sense of Presence while being in VR and its relation to User Experience therefore form crucial aspects, which are yet to be understood. This study aims at quantifying age and gender effects on this connection, involving 57 participants in VR, and performing a geocaching game using a mobile phone as experimental task to answer questionnaires measuring Presence (ITC-SOPI), User Experience (UEQ) and Usability (SUS). A higher Presence was found for the older participants, but there was no gender difference nor any interaction effects of age and gender. These findings are contractionary to preexisting limited work which has shown higher Presence for males and decreases of Presence with age. Four aspects discriminating this study from literature are discussed as explanations and as a starting point for future investigations into the topic. The results further showed higher ratings in favor of User Experience and lower ratings towards Usability for the older participants.
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Realidade Virtual , Masculino , Humanos , Inquéritos e QuestionáriosRESUMO
Realistic haptic feedback is a key for virtual reality applications in order to transition from solely procedural training to motor-skill training. Currently, haptic feedback is mostly used in low-force medical procedures in dentistry, laparoscopy, arthroscopy and alike. However, joint replacement procedures at hip, knee or shoulder, require the simulation of high-forces in order to enable motor-skill training. In this work a prototype of a haptic device capable of delivering double the force (35 N to 70 N) of state-of-the-art devices is used to examine the four most common haptic rendering methods (penalty-, impulse-, constraint-, rigid body-based haptic rendering) in three bimanual tasks (contact, rotation, uniaxial transition with increasing forces from 30 to 60 N) regarding their capabilities to provide a realistic haptic feedback. In order to provide baseline data, a worst-case scenario of a steel/steel interaction was chosen. The participants needed to compare a real steel/steel interaction with a simulated one. In order to substantiate our results, we replicated the study using the same study protocol and experimental setup at another laboratory. The results of the original study and the replication study deliver almost identical results. We found that certain investigated haptic rendering method are likely able to deliver a realistic sensation for bone-cartilage/steel contact but not for steel/steel contact. Whilst no clear best haptic rendering method emerged, penalty-based haptic rendering performed worst. For simulating high force bimanual tasks, we recommend a mixed implementation approach of using impulse-based haptic rendering for simulating contacts and combine it with constraint or rigid body-based haptic rendering for rotational and translational movements.
Assuntos
Artroplastia de Substituição , Interface Háptica , Humanos , Tecnologia Háptica , Artroscopia , Simulação por ComputadorRESUMO
Causal therapeutic approaches for amyloid diseases such as Alzheimer's and Parkinson's disease targeting toxic amyloid oligomers or fibrils are still emerging. Here, we show that theaflavins (TF1, TF2a, TF2b, and TF3), the main polyphenolic components found in fermented black tea, are potent inhibitors of amyloid-ß (Aß) and α-synuclein (αS) fibrillogenesis. Their mechanism of action was compared to that of two established inhibitors of amyloid formation, (-)-epigallocatechin gallate (EGCG) and congo red (CR). All three compounds reduce the fluorescence of the amyloid indicator dye thioflavin T. Mapping the binding regions of TF3, EGCG, and CR revealed that all three bind to two regions of the Aß peptide, amino acids 12-23 and 24-36, albeit with different specificities. However, their mechanisms of amyloid inhibition differ. Like EGCG but unlike congo red, theaflavins stimulate the assembly of Aß and αS into nontoxic, spherical aggregates that are incompetent in seeding amyloid formation and remodel Aß fibrils into nontoxic aggregates. When compared to EGCG, TF3 was less susceptible to air oxidation and had an increased efficacy under oxidizing conditions. These findings suggest that theaflavins might be used to remove toxic amyloid deposits.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Amiloide/química , Amiloide/metabolismo , Biflavonoides/farmacologia , Catequina/farmacologia , alfa-Sinucleína/metabolismo , Amiloide/efeitos dos fármacos , Peptídeos beta-Amiloides/química , Animais , Antioxidantes/farmacologia , Sítios de Ligação , Camellia sinensis/química , Catequina/análogos & derivados , Linhagem Celular Tumoral , Vermelho Congo/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Fluorescência , Interações Hidrofóbicas e Hidrofílicas , Placa Amiloide/tratamento farmacológico , Desnaturação Proteica/efeitos dos fármacos , RatosRESUMO
Epidemiological studies suggest that consumption of tomato products reduces the risk of CVD via antioxidant, hypocholesterolaemic and anti-inflammatory mechanisms. Although experimental data also describe beneficial effects on endothelial function, clinical data in human subjects are lacking. To test the hypothesis that tomato ingestion ameliorates endothelial function, we randomised healthy non-smoking postmenopausal women to consume a buttered roll with and without tomato purée (70 g) in a cross-over design. Endothelial-dependent flow-mediated dilation (FMD) and endothelial-independent nitro-mediated dilation of the brachial artery were assessed with high-resolution ultrasound (13 MHz linear array transducer). Acute (24 h) and long-term (7 d) effects were examined after daily consumption of the described meal. Nineteen volunteers completed the protocol and provided technically suitable ultrasound measurement data. Plasma lycopene levels increased from 0·30 (sem 0·04) (baseline) to 0·42 (sem 0·04) and to 0·74 (sem 0·06) µm after 24 h and 7 d, respectively, with tomato purée consumption. These data indicated an effective absorption of the tomato product. However, both acute and long-term tomato purée consumption had no effects on endothelium-dependent or -independent dilation of the brachial artery. In addition, we found no correlation between lycopene plasma levels and FMD. In conclusion, consumption of tomato products associated with a significant increase in plasma lycopene levels had no effects on endothelial function in healthy postmenopausal women.
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Doenças Cardiovasculares/prevenção & controle , Dieta , Endotélio Vascular/fisiologia , Solanum lycopersicum , Idoso , Artéria Braquial/diagnóstico por imagem , Artéria Braquial/fisiologia , Doenças Cardiovasculares/fisiopatologia , Carotenoides/sangue , Estudos Cross-Over , Endotélio Vascular/diagnóstico por imagem , Feminino , Humanos , Licopeno , Menopausa , Pessoa de Meia-Idade , Ultrassonografia , Vasodilatação/fisiologiaRESUMO
Orthopedic surgeons endure high physical stresses when performing surgery, as large forces and torques are applied commonly. Occupational risks are consequently higher when compared to other surgical disciplines. One example is the reaming of the acetabula during total hip arthroplasty, using customized instruments. This surgery may predispose the surgeon to overuse-related wrist pathology. In this study, torques acting along the reaming tool were measured, and the resulting forces applied to the orthopedic surgeons' wrists were estimated based on the measured torque data from hip reaming. Different reamer sizes and tool velocities were analyzed to determine how both parameters may influence the torques applied at the surgeon's wrist. Using a highly standardized setup, torques were measured while the reamer was pushed into the acetabula to remove cartilage. Maximum torques and stoppage torques at blocking of the reamer were compared between feed rates and reamer sizes. Peak values of the maximum torques along the reamer axis averaged 1.5-1.8 Nm. No significant difference between maximum torques and reamer sizes was found. A significant difference in maximum torques was noted between feed rates with a large effect (p = 0.010; η2 = 0.214) and a large interaction effect (p = 0.017; η2 = 0.186). Based on this experimental setup, it can be hypothesized that the impulsive behavior of the torque when the milling tool reaches the subchondral lamella could potentially contribute to wrist pathology. These preliminary data warrant further study. Consequently, torque limiters should be implemented in reamers to minimize the risk of occupation-related pathology to the wrist.
Assuntos
Artroplastia de Quadril , Cirurgiões Ortopédicos , Acetábulo/cirurgia , Humanos , Torque , PunhoRESUMO
OBJECTIVE: To examine whether treatment with epigallocatechin gallate (EGCG) influences progression of brain atrophy, reduces clinical and further radiologic disease activity markers, and is safe in patients with progressive multiple sclerosis (PMS). METHODS: We enrolled 61 patients with primary or secondary PMS in a randomized double-blind, parallel-group, phase II trial on oral EGCG (up to 1,200 mg daily) or placebo for 36 months with an optional open-label EGCG treatment extension (OE) of 12-month duration. The primary end point was the rate of brain atrophy, quantified as brain parenchymal fraction (BPF). The secondary end points were radiologic and clinical disease parameters and safety assessments. RESULTS: In our cohort, 30 patients were randomized to EGCG treatment and 31 to placebo. Thirty-eight patients (19 from each group) completed the study. The primary endpoint was not met, as in 36 months the rate of decrease in BPF was 0.0092 ± 0.0152 in the treatment group and -0.0078 ± 0.0159 in placebo-treated patients. None of the secondary MRI and clinical end points revealed group differences. Adverse events of EGCG were mostly mild and occurred with a similar incidence in the placebo group. One patient in the EGCG group had to stop treatment due to elevated aminotransferases (>3.5 times above normal limit). CONCLUSIONS: In a phase II trial including patients with multiple sclerosis (MS) with progressive disease course, we were unable to demonstrate a treatment effect of EGCG on the primary and secondary radiologic and clinical disease parameters while confirming on overall beneficial safety profile. CLINICALTRIALGOV IDENTIFIER: NCT00799890. CLASSIFICATION OF EVIDENCE: This phase II trial provides Class II evidence that for patients with PMS, EGCG was safe, well tolerated, and did not significantly reduce the rate of brain atrophy.
Assuntos
Catequina/análogos & derivados , Esclerose Múltipla Crônica Progressiva/tratamento farmacológico , Administração Oral , Adulto , Atrofia , Encéfalo/fisiopatologia , Catequina/farmacologia , Progressão da Doença , Método Duplo-Cego , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde , Distribuição AleatóriaRESUMO
OBJECTIVE: To assess the safety and efficacy of epigallocatechin-3-gallate (EGCG) add-on to glatiramer acetate (GA) in patients with relapsing-remitting multiple sclerosis (RRMS). METHODS: We enrolled patients with RRMS (aged 18-60 years, Expanded Disability Status Scale [EDSS] score 0-6.5), receiving stable GA treatment in a multicenter, prospective, double-blind, phase II, randomized controlled trial. Participants received up to 800 mg oral EGCG daily over a period of 18 months. The primary outcome was the proportion of patients without new hyperintense lesions on T2-weighted (T2w) brain MRI within 18 months. Secondary end points included additional MRI and clinical parameters. Immunologic effects of EGCG were investigated in exploratory experiments. RESULTS: A total of 122 patients on GA were randomly assigned to EGCG treatment (n = 62) or placebo (n = 60). We could not demonstrate a difference between groups after 18 months for the primary outcome or other radiologic (T2w lesion volume, T1w hypointense lesion number or volume, number of cumulative contrast-enhancing lesions, percent brain volume change), or clinical (EDSS, MS functional composite, and annualized relapse rate) parameter. EGCG treatment did not affect immune response to GA. Pharmacologic analysis revealed wide ranging EGCG plasma levels. The treatment was well tolerated with a similar incidence of mostly mild adverse events similar in both groups. CONCLUSION: In RRMS, oral EGCG add-on to GA was not superior to placebo in influencing MRI and clinical disease activity over 18 months. The treatment was safe at a daily dosage up to 800 mg EGCG. It did not influence immune parameters, despite indication of EGCG being bioavailable in patients. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that for patients with RRMS, EGCG added to GA did not significantly affect the development of new hyperintense lesions on T2-weighted brain MRI. TRIAL REGISTRATION INFORMATION: Clinical trial registration number: NCT00525668.
Assuntos
Catequina/análogos & derivados , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Adulto , Encéfalo/patologia , Catequina/uso terapêutico , Método Duplo-Cego , Acetato de Glatiramer/uso terapêutico , Humanos , Imageamento por Ressonância Magnética , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto JovemRESUMO
Improvement of endothelial function represents a major health effect of tea in humans. Ex vivo, tea and tea polyphenols stimulate nitric oxide (NO)-dependent vasodilation in isolated blood vessels. However, it was reported that polyphenols can generate reactive oxygen species (ROS) in vitro. We therefore aimed to elucidate the role of ROS production in tea polyphenol-induced vasodilation in explanted aortic rings. Vasorelaxation of rat aortic rings was assessed in an organ chamber model with low concentrations of epigallocatechin-3-gallate (EGCG), theaflavin-3,3'-digallate (TF3), and with green and black tea, with or without pretreatment with catalase or superoxide dismutase (SOD). The stability of EGCG and TF3 was measured by HPLC, and the levels of hydrogen peroxide (H2O2) were determined. EGCG and green tea-induced vasorelaxation was completely prevented by catalase and slightly increased by SOD. TF3 and black tea yielded similar results. Both EGCG and TF3 were rapidly degraded. This was associated with increasing H2O2 levels over time. Hydrogen peroxide concentrations produced in a time range compatible with tea polyphenol decay induced NO-dependent vasodilation in aortic rings. In conclusion, tea polyphenol-induced vasodilation in vitro is mediated by low levels of H2O2 generated during compound decay. The results could explain the apparent lack of vasodilatory effects of isolated tea polyphenols in humans.
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Total hip arthroplasty (THA) is a highly successful surgical procedure, but complications remain, including aseptic loosening, early dislocation and misalignment. These may partly be related to lacking training opportunities for novices or those performing THA less frequently. A standardized training setting with realistic haptic feedback for THA does not exist to date. Virtual Reality (VR) may help establish THA training scenarios under standardized settings, morphology and material properties. This work summarizes the development and acquisition of mechanical properties on hip reaming, resulting in a tissue-based material model of the acetabulum for force feedback VR hip reaming simulators. With the given forces and torques occurring during the reaming, Cubic Hermite Spline interpolation seemed the most suitable approach to represent the nonlinear force-displacement behavior of the acetabular tissues over Cubic Splines. Further, Cubic Hermite Splines allowed for a rapid force feedback computation below the 1 ms hallmark. The Cubic Hermite Spline material model was implemented using a three-dimensional-sphere packing model. The resulting forces were delivered via a human-machine-interaction certified KUKA iiwa robotic arm used as a force feedback device. Consequently, this novel approach presents a concept to obtain mechanical data from high-force surgical interventions as baseline data for material models and biomechanical considerations; this will allow THA surgeons to train with a variety of machining hardness levels of acetabula for haptic VR acetabulum reaming.
Assuntos
Acetábulo/cirurgia , Fenômenos Biomecânicos/fisiologia , Acetábulo/fisiologia , Artroplastia de Quadril , Simulação por Computador , Prótese de Quadril , Humanos , Realidade VirtualRESUMO
Epidemiological studies suggest that consumption of tea is associated with beneficial cardiovascular effects. Since different types of tea are consumed throughout the world, a question of much interest is whether green tea is superior to black tea in terms of cardiovascular protection. We therefore compared the effects of green and black tea on nitric oxide (NO) production and vasodilation and elucidated the tea compounds involved. We chose a highly fermented black tea and determined concentrations of individual tea compounds in both green and black tea of the same type (Assam). The fermented black tea was almost devoid of catechins. However, both teas stimulated eNOS activity and phosphorylation in bovine aortic endothelial cells (BAEC) as well as vasorelaxation in rat aortic rings to a similar extent. In green tea, only epigallocatechin-3-gallate (EGCG) resulted in pronounced NO production and NO-dependent vasorelaxation in aortic rings. During tea processing to produce black tea, the catechins are converted to theaflavins and thearubigins. Individual black tea theaflavins showed a higher potency than EGCG in NO production and vasorelaxation. The thearubigins in black tea are highly efficient stimulators of vasodilation and NO production. Green and black tea compounds induced comparable phosphorylation of eNOS and upstream signalling kinases. Whereas stimulation of eNOS activity by EGCG was only slightly affected by pretreatment of cells with various ROS scavengers, TF3(theaflavin-3',3-digallate)-induced eNOS activity was partially inhibited by PEG-catalase. These results implicate that highly fermented black tea is equally potent as green tea in promoting beneficial endothelial effects. Theaflavins and thearubigins predominantly counterbalance the lack of catechins in black tea. The findings may underline the contribution of black tea consumption in prevention of cardiovascular diseases.
Assuntos
Bebidas/estatística & dados numéricos , Catequina/análogos & derivados , Endotélio Vascular/fisiologia , Óxido Nítrico/metabolismo , Chá , Vasodilatação/fisiologia , Animais , Aorta/efeitos dos fármacos , Aorta/fisiologia , Encéfalo , Catequina/farmacologia , Bovinos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Humanos , Hidrocortisona/farmacologia , Óxido Nítrico Sintase Tipo III/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Extratos de Tecidos/farmacologiaRESUMO
Differences between men and women are being continuously identified in many human diseases. The underlying reasons are not yet fully understood. Beside the influence of endogenous hormones and life style, intrinsic sex-specific dimorphisms at the cellular level may also play a role. HUVECs from twin pairs of opposite sex provide an excellent tool to address the question of sex-specific differences at the molecular level. We compared for the first time protein levels of male and female HUVECs from dizygotic twins using a proteomic approach. To investigate differences under basal and stress conditions, cells were either left untreated or wounded and serum starved for different time points. Approximately 10% of all proteins monitored showed significant sexual dimorphisms in their level under the different conditions tested. The majority of the proteins displayed a higher abundance in female cells. The magnitude of the difference in protein levels between male and female cells was rather small. The most prominent differences throughout all conditions were observed for several X-chromosome encoded proteins with higher levels in female (UBA1, HDHD1) or in male cells (G6PD). Proteins involved in basic cellular processes, such as gene expression and translation (e.g. HMGN1, SRP54) displayed sex-specific levels in particular conditions only. SIGNIFICANCE: This study provides novel insights into sexual dimorphic protein levels in HUVECs from twin pairs of the opposite sex. The findings identify proteins with sex-specific differences in their levels under different cell culture conditions. The study also highlights the presence of X-chromosome encoded proteins escaping X-chromosomal inactivation. The results emphasize the need to consider the cellular sex of male and female HUVECs in in vitro experiments.
Assuntos
Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteoma/metabolismo , Proteômica , Caracteres Sexuais , Gêmeos Dizigóticos , Feminino , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Recém-Nascido , MasculinoRESUMO
A gas chromatography mass spectrometry (GC-MS) metabolomics protocol was modified for quenching, harvesting, and extraction of metabolites from adherent cells grown under high (20%) fetal calf serum conditions. The reproducibility of using either 50% or 80% methanol for quenching of cells was compared for sample harvest. To investigate the efficiency and reproducibility of intracellular metabolite extraction, different volumes and ratios of chloroform were tested. Additionally, we compared the use of total protein amount versus cell mass as normalization parameters. We demonstrate that the method involving 50% methanol as quenching buffer followed by an extraction step using an equal ratio of methanol:chloroform:water (1:1:1, v/v/v) followed by the collection of 6 mL polar phase for GC-MS measurement was superior to the other methods tested. Especially for large sample sets, its comparative ease of measurement leads us to recommend normalization to protein amount for the investigation of intracellular metabolites of adherent human cells grown under high (or standard) fetal calf serum conditions. To avoid bias, care should be taken beforehand to ensure that the ratio of total protein to cell number are consistent among the groups tested. For this reason, it may not be suitable where culture conditions or cell types have very different protein outputs (e.g., hypoxia vs. normoxia). The full modified protocol is available in the Supplementary Materials.
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Pyrrolizidine alkaloids (PA) are a group of secondary plant metabolites belonging to the most widely distributed natural toxins. PA intoxication of humans leads to severe liver damage, such as hepatomegaly, hepatic necrosis, fibrosis and cirrhosis. An acute consequence observed after ingestion of high amounts of PA is veno-occlusive disease (VOD) where the hepatic sinusoidal endothelial cells are affected. However, the mechanisms leading to VOD after PA intoxication remain predominantly unknown. Thus, we investigated PA-induced molecular effects on human umbilical vein endothelial cells (HUVEC). We compared the effects of PA with the effects of PA metabolites obtained by in vitro metabolism using liver homogenate (S9 fraction). In vitro-metabolized lasiocarpine and senecionine resulted in significant cytotoxic effects in HUVEC starting at 300⯵M. Initial molecular effect screening using a PCR array with genes associated with endothelial cell biology showed PA-induced upregulation of the Fas receptor, which is involved in extrinsic apoptosis, and regulation of a number of interleukins, as well as of different enzymes relevant for prostanoid synthesis. Modulation of prostanoid synthesis was subsequently studied at the mRNA and protein levels and verified by increased release of prostaglandin I2 as the main prostanoid of endothelial cells. All effects occurred only with in vitro-metabolically activated PA lasiocarpine and senecionine. By contrast, no effect was observed for the PA echimidine, heliotrine, lasiocarpine, senecionine, senkirkine and platyphylline in the absence of an external metabolizing system up to the highest tested concentration of 500⯵M. Overall, our results confirm the metabolism-dependent toxification of PA and elucidate the involved pathways. These include induction of inflammatory cytokines and deregulation of the prostanoid synthesis pathway in endothelial cells, linking for the first time PA-dependent changes in prostanoid release to distinct alterations at the mRNA and protein levels of enzymes of prostanoid synthesis.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Prostaglandinas/biossíntese , Alcaloides de Pirrolizidina/toxicidade , Animais , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Epoprostenol/metabolismo , Hepatopatia Veno-Oclusiva/induzido quimicamente , Células Endoteliais da Veia Umbilical Humana , Humanos , Inativação Metabólica/efeitos dos fármacos , Fígado/metabolismo , Masculino , Alcaloides de Pirrolizidina/farmacocinética , Ratos Wistar , Tromboxano A2/metabolismoRESUMO
BACKGROUND AND AIMS: Gonadal hormones are mainly thought to account for sex and gender differences in the incidence, clinical manifestation and therapy of many cardiovascular diseases. However, intrinsic sex differences at the cellular level are mostly overlooked. Here, we assessed sex-specific metabolic and functional differences between male and female human umbilical vein endothelial cells (HUVECs). METHODS: Cellular metabolism was investigated by bioenergetic studies (Seahorse Analyser) and a metabolomic approach. Protein levels were determined by Western blots and proteome analysis. Vascular endothelial growth factor (VEGF)-stimulated cellular migration was assessed by gap closure. HUVECs from dizygotic twin pairs were used for most experiments. RESULTS: No sex differences were observed in untreated cells. However, sexual dimorphisms appeared after stressing the cells by serum starvation and treatment with VEGF. Under both conditions, female cells had higher intracellular ATP and metabolite levels. A significant decline in ATP levels was observed in male cells after serum starvation. After VEGF, the ratio of glycolysis/mitochondrial respiration was higher in female cells and migration was more pronounced. CONCLUSIONS: These results point to an increased stress tolerance of female cells. We therefore propose that female cells have an energetic advantage over male cells under conditions of diminished nutrient supply. A more favourable energy balance of female HUVECs after serum starvation and VEGF could potentially explain their stronger migratory capacity.