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OBJECTIVE Over the last few years, there has been concern regarding the cardiovascular safety of selective cyclooxygenase (COX)-2 inhibitors and high-dose regimens of nonsteroidal anti-inflammatory drugs (NSAIDs). On the other hand, those compounds which elicit an almost complete (> 95%) and continuous suppression of platelet COX-1 may represent an exception. Apart from aspirin, which irreversibly inactivates COX-1, high-dose naproxen causes sustained COX-1 inhibition throughout the dose interval in some individuals. The present study examines whether naproxen sodium after a single dose administration and at steady state using "over-the-counter (OTC) doses" produces sufficient COX-1 inhibition. COX-2 inhibition was assessed concomitantly. METHODS Ex vivo inhibition of COX enzymes and the pharmacokinetics of naproxen were assessed in four volunteers receiving 220 mg naproxen sodium b.i.d. for 7 days. Blood samples were obtained pre-dose, at specified time points after the first dose on Day 1, and 12 hours after the previous evening dose on Days 2, 3, 4, 5 and 8. Recovery was assessed up to 36 hours after the last dose. Coagulation-induced thromboxane B2 formation and lipopolysaccharide-induced prostaglandin E2 synthesis were measured ex vivo in human whole blood as indices of COX-1 and COX-2 activity. RESULTS Maximal inhibition after a single dose and at steady state were as follows: 94% and 93% (COX-1), and 79% and 85% (COX-2). A greater than 95% COX-1 inhibition was observed transiently in 2 of 4 volunteers at the time of maximal plasma concentration after a single-dose administration and in 1 of 4 volunteers throughout the 12-hour dose interval at steady state. For both isoenzymes, COX inhibition correlated with naproxen plasma levels (ex vivo IC50 values of 35.48 micromol/l (COX-1) and 64.62 micromol/l (COX-2)). CONCLUSIONS Administration of naproxen sodium at OTC doses was associated with a profound inhibition of both COX enzymes. Although low-dose naproxen may elicit a virtually complete COX-1 inhibition in some individuals, it does not mimic the reliable, sustained and complete COX-1 blockade produced by aspirin. In conclusion, prolonged treatment with 220 mg naproxen sodium b.i.d. is not expected to provide sufficient cardioprotection in all patients, but may influence platelet function in some.

BACKGROUND AND AIM Diclofenac is a nonsteroidal antiinflammatory drug with potent analgesic and anti-inflammatory properties. An immediate-release formulation containing a low dose of 12.5 mg diclofenac-potassium (-K) is marketed as over the counter (OTC) medication in most European countries. An immediate-release formulation containing 25 mg diclofenac-K has now also been approved for OTC use. This study assessed the bioequivalence of two immediate-release diclofenac formulations when administered at the same dose. SUBJECTS AND METHODS A randomized, crossover, open-label study was conducted in 29 healthy volunteers to assess the bioequivalence of single 25 mg dose of diclofenac-K in two formulations: 2 x 12.5 mg as film-coated tablets and 1 x 25 mg as sugar-coated tablet. Blood samples for pharmacokinetic analyses were obtained over 10 hours post administration. RESULTS Plasma time courses of diclofenac were similar for the tested formulations. Mean AUC yen was 798 +/- 281 ng x h/ml (mean +/- SD) for the film-coated and 776 +/- 249 ng x h/ml for the sugar-coated formulation, respectively. The 2-sided 90% confidence interval for the mean test/reference ratio of AUCinfinity (95.5 - 107.5) fell within the predetermined equivalence range of 80 - 125%. Both formulations were rapidly absorbed; median time to maximal plasma concentration was 35 min in each group. Adverse events (peripheral erythema on the hand, headache, hypoesthesia) reported during the study were of mild severity and were considered as unlikely to be drug-related. CONCLUSION The two diclofenac-K immediate release formulations were pharmacokinetically similar. It can be concluded that the new sugar-coated tablet formulation is equivalent to the available film-coated tablet formulation with respect to the extent of diclofenac absorption.

Myofibroblasts promote tissue contractures during fibrotic diseases. To understand how spontaneous changes in the intracellular calcium concentration, [Ca2+](i), contribute to myofibroblast contraction, we analysed both [Ca2+](i) and subcellular contractions. Contractile events were assessed by tracking stress-fibre-linked microbeads and measured by atomic force microscopy. Myofibroblasts exhibit periodic (similar to 100 seconds) [Ca2+](i) oscillations that control small (similar to 400 nm) and weak (similar to 100 pN) contractions. Whereas depletion of [Ca2+](i) reduces these microcontractions, cell isometric tension is unaffected, as shown by growing cells on deformable substrates. Inhibition of Rho-and ROCK-mediated Ca2+-independent contraction has no effect on microcontractions, but abolishes cell tension. On the basis of this two-level regulation of myofibroblast contraction, we propose a single-cell lock-step model. Rho- and ROCK-dependent isometric tension generates slack in extracellular matrix fibrils, which are then accessible for the low-amplitude and high-frequency contractions mediated by [Ca2+](i). The joint action of both contraction modes can result in macroscopic tissue contractures of similar to 1 cm per month.

Lipocalin-2 (also known as NGAL) levels are elevated in obesity and diabetes yet relatively little is known regarding effects on the heart. We induced pressure overload (PO) in mice and found that lipocalin-2 knockout (LKO) mice exhibited less PO-induced autophagy and NLRP3 inflammasome activation than Wt. PO-induced mitochondrial damage was reduced and autophagic flux greater in LKO mice, which correlated with less cardiac dysfunction. All of these observations were negated upon adenoviral-mediated restoration of normal lipocalin-2 levels in LKO. Studies in primary cardiac fibroblasts indicated that lipocalin-2 enhanced priming and activation of NLRP3-inflammasome, detected by increased IL-1β, IL-18 and Caspase-1 activation. This was attenuated in cells isolated from NLRP3-deficient mice or upon pharmacological inhibition of NLRP3. Furthermore, lipocalin-2 induced release of HMGB1 from cells and NLRP3-inflammasome activation was attenuated by TLR4 inhibition. We also found evidence of increased inflammasome activation and reduced autophagy in cardiac biopsy samples from heart failure patients. Overall, this study provides new mechanistic insight on the detrimental role of lipocalin-2 in the development of cardiac dysfunction.

A novel imaging method employing the different kinetics of the energetic states of fluorescent markers is presented, where the contrast depends on the triplet kinetics. An attractive feature of triplet states is their long lifetime, rendering them highly sensitive to the immediate environment of their fluorescent host molecules. They typically have a lifetime of the order of ~10us to 10ms, which is 3-6 orders of magnitude longer than the lifetimes of fluorescent singlet states. The concept exploits characteristic variations of population of these states for different modulated excitations. It is fully compatible with low time- resolution detection by a CCD camera and does not impose any constraints on the concentration or the fluorescence brightness of the sample molecules to be measured. As first proof of principle, we demonstrate how triplet parameters of various fluorophores in different environments can be exploited for imaging. As control, the triplet parameters have been measured by FCS experiments. This concept opens for imaging of fluorophore markers in biological samples, as a novel contrast method for visualizing subtle changes in the immediate microenvironment of the fluorophore markers.

Strain devices with expandable polydimethylsiloxane (PDMS) culture membranes are frequently used to stretch cells in vitro, mimicking mechanically dynamic tissue environments. To immobilize cell-adhesive molecules to the otherwise non-adhesive PDMS substrate, hydrophobic, electrostatic and covalent surface coating procedures have been developed. The efficacy of different coating strategies to transmit stretches to cells however is poorly documented and has not been compared. We describe a novel and simple procedure to covalently bind extracellular matrix proteins to the surface of stretchable PDMS membranes. The method comprises PDMS oxygenation, silanization, and covalent protein cross-linking to the silane. We demonstrate improved attachment (∼2-fold), spreading (∼2.5-fold) and proliferation (∼1.2-fold) of fibroblasts to our new coating over established coating procedures. Further, we compared the efficiency of different PDMS coating techniques to transmit stretches. After 15% stretch, the number of maximally (15 ± 5%) stretched cells on our PDMS surface coating was ∼7-fold higher compared with alternative coating protocols. Hence, covalent linkage of adhesive molecules is superior to non-covalent methods in providing a coating that resists large deformations and that fully transmit this stretch to cultured cells. © 2008 Elsevier Ltd. All rights reserved.

Myofibroblasts are reparative connective tissue cells that contribute to the reconstruction of injured tissue by secreting new extracellular matrix and by exerting high contractile force. Deregulation of these activities results in tissue contracture and development of fibrosis which makes the myofibroblast an important target for anti-fibrotic therapies. Two principle factors drive the development of myofibroblasts from different precursor cells and guarantee maintenance of the contractile phenotype: mechanical stress and transforming growth factor beta (TGFβ1). In this mini-review, we recapitulate the current understanding (1) of how myofibroblasts feel stress using specialized matrix adhesions, (2) of the level of stress that is required to induce their development and (3) of how myofibroblast mechanical activity can have a direct influence on the level of TGFβ1 activation. From these findings it emerges that the specific matrix adhesion structures of myofibroblasts are promising targets to modulate myofibroblast differentiation and activity. © 2008 Elsevier Ltd. All rights reserved.

We present a functional imaging method based on triplet state kinetics. The triplet state of many known fluorophores show a triplet lifetime typically in the order of ~10e-6 – 10e-3 s, 3-6 orders of magnitude longer than the associated fluorescence singlet lifetime. As a consequence triplet state sensing is more exposed to the microenvironment of fluorophores and can be used as a functional molecular antenna. Using an adaptive modulated excitation scheme, these long living triplet state dynamics are accessible by standard CCD detector technology. We built an imaging setup allowing extraction of triplet lifetime maps. Effective correction of the competitive bleaching component is achieved by simultaneous monitoring of bleaching. The signal processing is based on a linear regressor analysis enabling extraction of the triplet state lifetime. As a first proof of principle, we investigated oxygen as a triplet state quencher on a sample of Tetramethylrhodamine (TMR) printed on a glass cover slide by soft lithography. First bioassays will be presented indicating the high potential for functional and metabolic imaging in life cell applications.

The present study aimed to (1) employ the method for evaluation of vibration containing multiple shocks according to ISO/CD 2631-5:2014 (Model 1) and DIN SPEC 45697:2012 in a cohort of 537 professional drivers, (2) deliver the results for a re-analysis of epidemiological data obtained in the VIBRISKS study, (3) clarify the extent to which vibration acceleration and individual variables influence risk values, such as the daily compressive dose S(ed) and the risk factor R, and (4) compare the results with in vivo measurements and those obtained in previous studies with similar models. The risk factor R was influenced by the acceleration, lifetime exposure duration, sitting posture, age at the start of exposure and body mass/body mass index in order of decreasing effect. Age and annual and daily exposure duration had only a marginal effect. The daily compressive dose S(ed) and the risk factor R showed weak linear association with the daily vibration exposure A(8) and the vibration dose value VDV. The study revealed high shear forces in the lumbar spine.In a re-analysis of an epidemiological study of professional drivers, a software tool available with standards DIN SPEC 45697:2012 and ISO/CD 2631–5:2014 Model 1 was used to calculate the risk to the lumbar spine in terms of daily compressive dose S(ed) and risk factor R. The tool was found to be suitable for risk assessment in a large cohort.