Perceived realism of haptic rendering methods for bimanual high force tasks: original and replication study.

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.

Pazopanib with 5-FU and oxaliplatin as first line therapy in advanced gastric cancer: A randomized phase-II study-The PaFLO trial. A study of the Arbeitsgemeinschaft Internistische Onkologie AIO-STO-0510.

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.

Wrist at risk? - Considerations derived from a novel experimental setup to assess torques during hip reaming with potential implications on the orthopedic surgeons' health.

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.

Vasodilation of Tea Polyphenols Ex Vivo Is Mediated by Hydrogen Peroxide Under Rapid Compound Decay.

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.

Modified Protocol of Harvesting, Extraction, and Normalization Approaches for Gas Chromatography Mass Spectrometry-Based Metabolomics Analysis of Adherent Cells Grown Under High Fetal Calf Serum Conditions.

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.

Sex-specific metabolic and functional differences in human umbilical vein endothelial cells from twin pairs.

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.

Pyrrolizidine alkaloid-induced alterations of prostanoid synthesis in human endothelial cells.

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 µM. 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 µM. 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.

A Possible Indicator of Oxidative Damage in Smokers: (13Z)-Lycopene?

In vitro, the gaseous phase of cigarette smoke is known to induce both isomerization and degradation of dietary carotenoids, such as ß-carotene and lycopene. However, the effects of cigarette smoke on the composition of circulating lycopene in vivo are not well understood. In this study, we examined the lycopene profiles of plasma from non-smokers and smokers. No oxidative intermediates of lycopene that have been observed previously in vitro were detected in the plasma, but evidence of isomerization of the carotenoid was seen. Four geometric forms of lycopene were detected in the plasma of both smokers and non-smokers, namely the (5Z), (9Z), (13Z) and (all-E) forms. The relative amounts of these isomers differed between the two cohorts and there was a significant difference (p < 0.05) between smokers and non-smokers for the ratio of total-Z:all-E lycopene, and in the relative amounts of (13Z) and (all-E)-lycopene. The ratio of (all-E):(13Z)-lycopene was 0.84:1.00 in smokers compared to 1.04:1.00 in non-smokers. In smokers, the (13Z)-isomer was generated in preference to the more thermodynamically stable (5Z) and (9Z)-isomers. This mirrors the scenario seen in vitro, in which the formation of (13Z)-lycopene was the main isomer that accompanied the depletion of (all-E) lycopene, when exposed to cigarette smoke. The results suggest that the relative amount of (13Z)-lycopene could be used as an indicator of oxidative damage to lycopene in vivo.

Metabolic response to epigallocatechin-3-gallate in relapsing-remitting multiple sclerosis: a randomized clinical trial.

BACKGROUND: Muscle weakness and fatigue are common symptoms in multiple sclerosis (MS). Green tea catechins such as (-)epigallocatechin-3-gallate (EGCG) are known to improve energy metabolism at rest and during exercise. OBJECTIVE: We tested the hypothesis that EGCG improves energy metabolism and substrate utilization in patients with MS. DESIGN: Eighteen patients (8 men) with relapsing-remitting MS (expanded disability status scale score <4.5, all receiving glatiramer acetate) participated in this randomized, double-blind, placebo-controlled, crossover trial at a clinical research center. All patients received EGCG (600 mg/d) and placebo over 12 wk (4-wk washout in between). After each intervention, fasting and postprandial energy expenditure (EE), as well as fat oxidation (FAOx) and carbohydrate oxidation (CHOx) rates, were measured either at rest or during 40 min of exercise (0.5 W/kg). At rest, blood samples and microdialysates from adipose tissue and skeletal muscle were also taken. RESULTS: At rest, postprandial EE and CHOx, as well as adipose tissue perfusion and glucose supply, were significantly lower in men but higher in women receiving EGCG compared with placebo. During exercise, postprandial EE was lower after EGCG than after placebo, indicating an increased working efficiency (men > women). After placebo, exercise EE was mainly fueled by FAOx in both men and women. After EGCG, there was a shift to a higher and more stable CHOx during exercise in men but not in women. CONCLUSIONS: Our data indicate that EGCG given to patients with MS over 12 wk improves muscle metabolism during moderate exercise to a greater extent in men than in women, possibly because of sex-specific effects on autonomic and endocrine control.

LXR agonism improves TNF-α-induced endothelial dysfunction in the absence of its cholesterol-modulating effects.

Stimulation of the liver X receptor (LXR) is associated with anti-inflammatory and vascular-protective effects under hyperlipemic conditions. We examined whether LXR stimulation influences TNF-α-induced endothelial dysfunction under normolipemic conditions. Endothelium-dependent vasorelaxation of aortic rings was determined in an organ water bath. Human umbilical vein endothelial cells (HUVEC) were exposed to TNF-α (10 ng/ml) in the presence or absence of 5 µM of the LXR agonist T0901317 or GW3965 and changes in TNF-α-induced endothelial cell apoptosis, inflammation, oxidative stress, and NO metabolism were analyzed. T0901317 improved TNF-α-impaired endothelium-dependent relaxation of aortic rings in response to acetylcholine. T0901317 decreased the TNF-α-induced apoptosis and inflammation as indicated by a decrease in caspase 3/7 activity, VCAM-1 mRNA expression and subsequent mononuclear cell adhesion. Furthermore, T0901317 reduced the expression of the oxidative stress markers: AT1R, NOX4, and p22phox and normalized the TNF-α-induced NOX activity to basal levels. In line with the reduced AT1R expression, T0901317 impaired the Ang II responsiveness. T0901317 influenced NO metabolism as indicated by a decrease in TNF-α-upregulated arginase activity, a reversal of TNF-α-induced downregulation of argininosuccinate synthase mRNA expression and eNOS expression to basal levels and a raise in NO production. Furthermore, T0901317 decreased the TNF-α-induced superoxide and nitrotyrosine production, but did not upregulate the TNF-α-downregulated eNOS dimer/monomer ratio. Silencing of LXRß, but not of LXRα, abrogated the anti-apoptotic effects of T0901317. We conclude that LXR agonism improves TNF-α-impaired endothelial function via its anti-apoptotic, anti-inflammatory, and anti-oxidative properties and its capacity to restore TNF-α-impaired NO bioavailability independent of its cholesterol-modulating effects.

Cellular targets for the beneficial actions of tea polyphenols.

Green and black teas contain different biologically active polyphenolic compounds that might offer protection against a variety of human diseases. Although promising experimental and clinical data have shown protective effects, limited information is available on how these beneficial effects of tea polyphenols are mediated at the cellular level. Evidence is accumulating that catechins in green tea as well as theaflavins and thearubigins from black tea are the substances responsible for the physiologic effects of tea in vitro. The green tea catechin epigallocatechin-3-gallate (EGCG) is generally considered to be the biologically most active compound in vitro. The changes in the activities of various protein kinases, growth factors, and transcription factors represent a common mechanism involved in cellular effects of tea polyphenols. In addition to modification of intracellular signaling by activation of cellular receptors, it was shown that, at least for EGCG, tea polyphenols can enter the cells and directly interact with their molecular targets within cells. There, they frequently result in opposite effects in primary compared with tumor cells. Although tea polyphenols were long regarded as antioxidants, research in recent years has uncovered their prooxidant properties. The use of high nonphysiologic concentrations in many cell culture studies raises questions about the biological relevance of the observed effects for the in vivo situation. Efforts to attribute functional effects in vivo to specific molecular targets at the cellular level are still ongoing.

Black tea theaflavins inhibit formation of toxic amyloid-ß and α-synuclein fibrils.

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.

Lack of effects of tomato products on endothelial function in human subjects: results of a randomised, placebo-controlled cross-over study.

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.

Proteasome inhibition prevents experimentally-induced endothelial dysfunction.

AIMS: We recently demonstrated that non-toxic inhibition of the proteasome upregulates antioxidative enzymes and leads to an adaptive transcriptional pattern in endothelial cells. We therefore hypothesized that proteasome inhibition could prevent experimentally-induced endothelial dysfunction. As there are conflicting data about the effects of proteasome inhibition on endothelial function, we investigated whether proteasome inhibition could prevent experimentally-induced endothelial dysfunction. MAIN METHODS: Endothelial dysfunction in isolated rat aortic rings was induced by incubation of rings with TNFalpha for 48 h. To study the effects of the inhibition of the proteasome, selected rings were co-treated with proteasome inhibitors. Vasorelaxation and expression of genes involved in endothelial function were evaluated. KEY FINDINGS: Incubation of rat aortic rings with TNFalpha for 48 h led to significant dose-dependent reduction of acetylcholine-induced vasorelaxation. Co-incubation with TNFalpha and the proteasome inhibitor MG132 resulted in dose-dependent improvement of endothelium-dependent vasorelaxation in comparison to rings treated with TNFalpha alone. Levels of eNOS mRNA and protein were reduced despite improved vascular function after treatment with MG132. MG132 markedly suppressed mRNA levels of NADPH oxidase subunits and increased SOD1 expression. Superoxide production was reduced in rings incubated with MG132 in comparison to controls. TNFalpha-induced upregulation of the potent vasoconstrictor endothelin was abolished by MG132. SIGNIFICANCE: Proteasome inhibition prevents TNFalpha-induced vascular dysfunction by reduction of superoxide production and suppression of endothelin levels. The balance between vasoconstriction and vasodilatation is shifted in favour of endothelium-dependent vasodilation.

Characteristics of catechin- and theaflavin-mediated cardioprotection.

Catechins and theaflavins-the main polyphenolic substances of green and black tea, respectively-exert a plethora of beneficial effects on the cardiovascular system. In a model of H(2)O(2)-mediated oxidative stress, we investigated the effects of epigallocatechin-3-gallate (EGCG) and theaflavin-3,3'-digallate (TF3) on neonatal rat cardiomyocytes. Pretreatment with EGCG or TF3 1 hr prior to induction of oxidative stress by H(2)O(2) effectively protected cardiac myocytes as determined by measuring release of lactate dehydrogenase after 24 hrs. Longer pre-incubation times resulted in significant loss of protection. To enable further mechanistic insight, we investigated expression of antioxidative enzymes and activation of prosurvival signaling cascades. Whereas mRNA levels of glutathione peroxidase 3, superoxide dismutase 1, and catalase were not influenced by both polyphenols, heme oxygenase (HO-1) was selectively upregulated by EGCG-but not by TF3. However, inhibition of HO-1 did not diminish polyphenol-mediated cardioprotection. While EGCG and TF3 activated Akt, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase, inhibition of these kinases did not attenuate polyphenol-mediated protection. Loading of cardiomyocytes with dichlorofluorescein revealed that intracellular levels of reactive oxygen species were significantly reduced after treatment with EGCG or TF3 as early as 30 mins after induction of oxidative stress. In conclusion, activation of prosurvival signaling kinases and upregulation of antioxidative enzymes do not play a major role in tea polyphenol-mediated cardioprotection.

Molecular targets of tea polyphenols in the cardiovascular system.

Tea-derived polyphenols have attracted considerable attention in the prevention of cancer and cardiovascular diseases. In comparison to tumour cells, the elucidation of their molecular targets in cardiovascular relevant cells is still at the beginning. Although promising experimental and clinical data demonstrate protective effects for the cardiovascular system, little information is actually available on how these beneficial effects of tea polyphenols are mediated at the cellular level. By affecting the activity of receptor and signal transduction kinases, both catechins and theaflavins--the major ingredients of green and black tea, respectively--exert a variety of cardiovascular beneficial effects. In general, the number and positions of galloyl groups have major influence on the potency of polyphenols. Compared to their broad impact on cellular signal transduction, tea polyphenols reveal little transcriptional effects. However, more detailed and profound analysis of molecular actions in different cells of the cardiovascular system is necessary before safe clinical use of tea polyphenols for treatment of cardiovascular diseases will become possible.

Nontoxic proteasome inhibition activates a protective antioxidant defense response in endothelial cells.

Inhibitors of the ubiquitin-proteasome system offer a new and promising approach in the therapy of proliferative and inflammatory diseases. In order to narrow the therapeutic window for cytotoxic effects on the one hand and nontoxic, anti-inflammatory effects on the other hand, we elucidated the complex cellular effects of toxic versus nontoxic proteasome inhibition in human endothelial cells by expressional profiling. Nontoxic doses of proteasome inhibitors induced a defined, dose-dependent transcriptional response that was markedly attenuated in terms of gene number and amplitude of regulation compared to toxic doses. In particular, we observed uniform upregulation of several antioxidative enzymes and differential regulation of genes involved in endothelial function. This adaptive transcriptional pattern was translated into a protective response of endothelial cells against H(2)O(2)-induced oxidative stress and into improvement of endothelial function of rat aortic rings. Our data thus suggest that nontoxic proteasome inhibition might offer a new therapeutic approach for the treatment of endothelial dysfunction in cardiovascular disorders.

The flavonoid phloretin suppresses stimulated expression of endothelial adhesion molecules and reduces activation of human platelets.

Atherosclerosis is a chronic inflammatory disease accompanied by the expression of endothelial adhesion molecules. Phloretin is a plant-derived phytochemical that is mainly present in apples. Because phloretin is reported to promote antioxidative activities, we investigated the effects of phloretin on cytokine-induced expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) in human umbilical vein endothelial cells (HUVECs). Phloretin prevented TNF-alpha-stimulated upregulation of VCAM-1, ICAM-1, and E-selectin expression in a concentration-dependent manner. To the same extent as for TNF-alpha, phloretin also inhibited IL-1beta-induced upregulation in expression of all 3 adhesion molecules. Inhibition of cytokine-induced adhesion molecule expression for VCAM-1, ICAM-1, and E-selectin was detected already at the level of mRNA. Preincubation with phloretin dose-dependently attenuated TNF-alpha-stimulated adhesion of monocytic THP-1 cells to HUVECs and human aortic endothelial cells. Phloretin did not affect TNF-alpha-stimulated activation of nuclear factor kappaB (NF-kappaB) but inhibited activation of interferon regulatory factor 1, a transcription factor involved in the regulation of endothelial cell adhesion molecule expression. In human platelets, phloretin diminished adenosine diphosphate (ADP) and thrombin receptor-activating peptide-stimulated expression of the activated form of the GPIIb/IIIa complex and reduced platelet aggregation stimulated by ADP. Thus phloretin may have beneficial effects in the onset and progression of cardiovascular diseases.

The tea flavonoid epigallocatechin-3-gallate reduces cytokine-induced VCAM-1 expression and monocyte adhesion to endothelial cells.

Attachment of leukocytes to the vascular endothelium and the subsequent migration of cells into the vessel wall are early events in atherogenesis. This process requires the expression of endothelial adhesion molecules. Since tea catechins are reputed to promote antiatherogenic activities, we investigated the effects of various tea catechins-i.e., epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC), and epigallocatechin-3-gallate (EGCG)-on cytokine-induced expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) in HUVECs by ELISA. EGCG and to a lesser extent ECG prevented the induction of VCAM-1 expression in a concentration-dependent manner after stimulation with TNF-alpha, whereas EC and EGC were without effect. EGCG also inhibited the IL-1beta-induced induction of VCAM-1 expression. Inhibition of cytokine-induced VCAM-1 expression was manifested already on the transcriptional level. Furthermore, EGCG reduced the TNF-alpha-induced adhesion of THP-1 cells to HUVECs. EGCG did not influence TNF-alpha-stimulated NF-kappaB activation.

Long-term up-regulation of eNOS and improvement of endothelial function by inhibition of the ubiquitin-proteasome pathway.

The ubiquitin-proteasome system is the major pathway for intracellular protein degradation in eukaryotic cells. Endothelial nitric oxide synthase (eNOS) is the key enzyme of vascular homeostasis involved in the pathophysiology of several cardiovascular diseases. The aim of our study was to investigate whether eNOS expression and activity are regulated by the proteasome. Bovine pulmonary artery endothelial cells (CPAE cells) were treated with the proteasome inhibitor MG132. MG132 (50-250 nmol/L) dose-dependently increased mRNA and protein levels of eNOS. Comparable results were obtained with other specific proteasome inhibitors, whereas the nonproteasomal calpain and cathepsin inhibitor ALLM had no effect. Efficacy of proteasome inhibition was evidenced by accumulation of poly-ubiquitinylated proteins and by measuring proteasomal activity in cell extracts. Cycloheximide prevented up-regulation of eNOS protein, indicating that post-translational stabilization of eNOS is not involved. eNOS activity was increased up to 2.8-fold (MG132 100 nmol/L, 48 h). Incubation of rat aortic rings with MG132 significantly enhanced endothelial-dependent vasorelaxation. Single MG132 treatment (100 nmol/L) induced long-term effects in CPAE cells, with increases of eNOS protein and activity for up to 10 days. Our results indicate that low-dose proteasome inhibition enhances eNOS expression and activity, and improves endothelial function.