Isoform-specific regulation of the actin-organizing protein palladin during TGF-beta1-induced myofibroblast differentiation.

Contractile myofibroblasts are responsible for remodeling of extracellular matrix during wound healing; however, their continued activity results in various fibrocontractive diseases. Conversion of fibroblasts into myofibroblasts is induced by transforming growth factor-beta1 (TGF-beta1) and is hallmarked by the neo-expression of alpha-smooth muscle actin (alpha-SMA), a commonly used myofibroblast marker. Moreover, myofibroblast differentiation and acquisition of the contractile phenotype involves functionally important alterations in the expression of actin-organizing proteins.

A validated high-performance liquid chromatographic assay for determination of lumiracoxib in human plasma.

Lumiracoxib {2-[(2-fluoro-6-chlorophenyl)amino]-5-methyl-benzeneacetic acid} is a highly selective and potent cyclooxygenase-2 (COX-2) inhibitor, which is chemically distinct from other coxibs in that it contains a carboxylic group and is weakly acidic. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of lumiracoxib in human plasma. The analyte was separated on a reversed-phase column with acetonitrile and 0.

Shear stress modulates the expression of thrombospondin-1 and CD36 in endothelial cells in vitro and during shear stress-induced angiogenesis in vivo.

Binding of thrombospondin-1 (TSP-1) to the CD36 receptor inhibits angiogenesis and induces apoptosis in endothelial cells (EC). Conversely, matrix-bound TSP-1 supports vessel formation. In this study we analyzed the shear stress-dependent expression of TSP-1 and CD36 in endothelial cells in vitro and in vivo to reveal its putative role in the blood flow-induced remodelling of vascular networks.

Masters and servants of the force: the role of matrix adhesions in myofibroblast force perception and transmission.

Fibroblast-to-myofibroblast differentiation - a key event in the development of fibrocontractive diseases and in wound granulation tissue contraction - is hallmarked by the formation of stress fibers and the neo-expression of alpha-smooth muscle actin. Incorporation of the smooth muscle actin isoform into stress fibers confers to myofibroblasts a high contractile activity which is transmitted to the extracellular matrix at sites of specialized adhesions, termed 'fibronexus' in tissue and 'supermature focal adhesions' in two-dimensional cell culture. Myofibroblast differentiation requires a mechanically restrained environment in conjunction with the action of growth factors like transforming growth factor beta and specialized matrix molecules like the ED-A splice variant of fibronectin.

Biocompatibility of bioresorbable poly(L-lactic acid) composite scaffolds obtained by supercritical gas foaming with human fetal bone cells.

The aim of this investigation was to test the biocompatibility of three-dimensional bioresorbable foams made of poly(L-lactic acid) (PLA), alone or filled with hydroxyapatite (HA) or beta-tricalcium phosphate (beta-TCP), with human primary osteoblasts, using a direct contact method. Porous constructs were processed by supercritical gas foaming, after a melt-extrusion of ceramic/polymer mixture. Three neat polymer foams, with pore sizes of 170, 310, and 600 microm, and two composite foams, PLA/5 wt% HA and PLA/5 wt% beta-TCP, were examined over a 4-week culture period.

Focal adhesion size controls tension-dependent recruitment of alpha-smooth muscle actin to stress fibers.

Expression of alpha-smooth muscle actin (alpha-SMA) renders fibroblasts highly contractile and hallmarks myofibroblast differentiation. We identify alpha-SMA as a mechanosensitive protein that is recruited to stress fibers under high tension. Generation of this threshold tension requires the anchoring of stress fibers at sites of 8-30-microm-long "supermature" focal adhesions (suFAs), which exert a stress approximately fourfold higher (approximately 12 nN/microm2) on micropatterned deformable substrates than 2-6-microm-long classical FAs.

More pronounced inhibition of cyclooxygenase 2, increase in blood pressure, and reduction of heart rate by treatment with diclofenac compared with celecoxib and rofecoxib.

Objective: Recent findings suggest that permanent blockade of cyclooxygenase 2 (COX-2) is one factor contributing to the cardiovascular side effects of selective COX-2 inhibitors (coxibs) and nonsteroidal antiinflammatory drugs (NSAIDs). The present study compared the extent and time course of COX-2 inhibition and the effects on cardiovascular parameters (changes in blood pressure and heart rate) between various antirheumatic doses of diclofenac, celecoxib, and rofecoxib in healthy elderly volunteers.

Methods: A randomized, parallel-group study was conducted in volunteers receiving 75 mg diclofenac twice daily, 200 mg celecoxib twice daily, or 25 mg rofecoxib once daily for 8 days.

R(+)-methanandamide and other cannabinoids induce the expression of cyclooxygenase-2 and matrix metalloproteinases in human nonpigmented ciliary epithelial cells.

Prostaglandins (PGs) and matrix metalloproteinases (MMP) have been implicated in lowering intraocular pressure (IOP) by facilitating aqueous humor outflow. A possible role of cyclooxygenase-2 (COX-2) in this process was emphasized by findings showing an impaired COX-2 expression in the nonpigmented ciliary epithelium (NPE) of patients with primary open-angle glaucoma. Using human NPE cells, the present study therefore investigated the effect of the IOP-lowering cannabinoid R(+)-methanandamide [R(+)-MA] on the expression of COX-2 and different MMPs and tissue inhibitors of MMPs (TIMPs).

R(+)-methanandamide elicits a cyclooxygenase-2-dependent mitochondrial apoptosis signaling pathway in human neuroglioma cells.

Purpose: Cannabinoids have been associated with tumor regression and apoptosis of cancer cells. Recently, we have shown that R(+)-methanandamide (R(+)-MA) induces apoptosis of H4 human neuroglioma cells via a mechanism involving de novo expression of the cyclooxygenase-2 (COX-2) enzyme. The present study investigated a possible involvement of a mitochondrial-driven pathway in this process.

Prostaglandin E2 induces cyclooxygenase-2 expression in human non-pigmented ciliary epithelial cells through activation of p38 and p42/44 mitogen-activated protein kinases.

Prostaglandins (PGs) have been implicated in lowering intraocular pressure (IOP). A possible role of cyclooxygenase-2 (COX-2) in this process was emphasized by findings showing impaired COX-2 expression in the non-pigmented ciliary epithelium (NPE) of patients with primary open-angle glaucoma. The present study investigates the effect of the major COX-2 product, PGE(2), on the expression of its synthesizing enzyme in human NPE cells (ODM-2).

Determination of the endocannabinoid anandamide in human plasma by high-performance liquid chromatography.

Anandamide (N-arachidonylethanolamine) is an endogenous cannabinoid receptor ligand that has been implicated in various physiological and pathophysiological functions. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with fluorometric detection was validated and applied for the analysis of anandamide in human plasma. Following derivatization with the fluorogenic reagent 4-(N,N-dimethylaminosulfonyl)-7-(N-chloroformylmethyl-N-methyl-amino)-2,1,3-benzoxadiazole (DBD-COCl), the analyte was separated using an acetonitrile-water gradient at a flow rate of 0.

Interstitial fluid flow induces myofibroblast differentiation and collagen alignment in vitro.

The differentiation of fibroblasts to contractile myofibroblasts, which is characterized by de novo expression of alpha-smooth muscle actin (alpha-SMA), is crucial for wound healing and a hallmark of tissue scarring and fibrosis. These processes often follow inflammatory events, particularly in soft tissues such as skin, lung and liver. Although inflammatory cells and damaged epithelium can release transforming growth factor beta1 (TGF-beta1), which largely mediates myofibroblast differentiation, the biophysical environment of inflammation and tissue regeneration, namely increased interstitial flow owing to vessel hyperpermeability and/or angiogenesis, may also play a role.

Wound-healing defect of CD18(-/-) mice due to a decrease in TGF-beta1 and myofibroblast differentiation.

We studied the mechanisms underlying the severely impaired wound healing associated with human leukocyte-adhesion deficiency syndrome-1 (LAD1) using a murine disease model. In CD18(-/-) mice, healing of full-thickness wounds was severely delayed during granulation-tissue contraction, a phase where myofibroblasts play a major role. Interestingly, expression levels of myofibroblast markers alpha-smooth muscle actin and ED-A fibronectin were substantially reduced in wounds of CD18(-/-) mice, suggesting an impaired myofibroblast differentiation.

Area-specific effects of brain-derived neurotrophic factor (BDNF) genetic ablation on various neuronal subtypes of the mouse brain.

The effects of brain-derived neurotrophic factor (BDNF) on the development of presynaptic terminals and of neuronal subtypes in various brain areas were studied in BDNF-knockout (BDNF-/-) mice at postnatal days 15-17. Western analysis revealed no changes in the overall amount of a variety of synaptic proteins in BDNF-/- mice as compared to wild type mice. In addition, the complex between the vesicular proteins, synaptophysin and synaptobrevin, as well as their respective homodimers were unaltered.

Latanoprost induces matrix metalloproteinase-1 expression in human nonpigmented ciliary epithelial cells through a cyclooxygenase-2-dependent mechanism.

Prostaglandins (PGs) have been implicated in the regulation of intraocular pressure (IOP) by facilitating the remodeling of tissues involved in aqueous humor outflow. A contribution of cyclooxygenase-2 (COX-2)-dependent PGs to this process was emphasized by a recent study showing an impaired COX-2 expression in the nonpigmented ciliary epithelium (NPE) of patients with primary open-angle glaucoma. With the use of human NPE cells (ODM-2), the present study therefore investigated the effect of the antiglaucomatous drug latanoprost (PGF2alpha analog) on the expression of COX-2 and its association with the induction of matrix metalloproteinases (MMPs).

Comparison of stereoscopic digital imaging and slide film photography in the identification of macular degeneration.

Background: This study compared the sensitivity and specificity of stereoscopic digital photography of the retina through a dilated pupil with a 45 degrees nonmydriatic camera and Joint Photographic Experts Group (JPEG) compression of the images with the sensitivity and specificity of 35-mm slide film photography in the identification of age-related macular degeneration (AMD).

Methods: Consecutive patients with a diagnosis of AMD were enrolled. Stereoscopic retinal images of the disc, macula and temporal macula were captured with a digital 45 degrees nonmydriatic camera (then compressed into JPEG format) and with a standard fundus camera and slide film.

Simultaneous determination of oxycodone and its major metabolite, noroxycodone, in human plasma by high-performance liquid chromatography.

Oxycodone (14-hydroxy-7,8-dihydrocodeinone) is a potent opioid receptor agonist. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of oxycodone and its major metabolite, noroxycodone, in human plasma. The analytes were separated using a mobile phase, consisting of acetonitrile and phosphate buffer (8:92, v/v) at a flow rate of 1 mL/min, and UV detection at 205 nm.

The N-terminal Ac-EEED sequence plays a role in alpha-smooth-muscle actin incorporation into stress fibers.

We have previously shown that the N-terminal sequence AcEEED of alpha-smooth-muscle actin causes the loss of alpha-smooth-muscle actin from stress fibers and a decrease in cell contractility when introduced in myofibroblasts as a cell-penetrating fusion peptide. Here, we have investigated the function of this sequence on stress fiber organization in living cells, using enhanced green fluorescent protein (EGFP)-tagged alpha-smooth-muscle actin. The fusion peptide provokes the gradual disappearance of EGFP fluorescence of alpha-smooth-muscle actin from stress fibers and the formation of hitherto unknown rod-like structures.

JPEG compression of stereoscopic digital images for the diagnosis of diabetic retinopathy via teleophthalmology.

Background: Canada's vast size and remote rural communities represent a significant hurdle for successful monitoring and evaluation of diabetic retinopathy. Teleophthalmology may provide a solution to overcome this problem. We investigated the application of Joint Photographic Experts Group (PEG) compression to digital retinal images to determine whether JPEG compression could reduce file sizes while maintaining sufficient quality and detail to accurately diagnose diabetic retinopathy.

Bioavailability of diclofenac potassium at low doses.

Aim: Diclofenac-K has been recently launched at low oral doses in different countries for over-the-counter use. However, given the considerable first-pass metabolism of diclofenac, the degree of absorption of diclofenac-K at low doses remained to be determined. The aim of this study was to determine the bioavailability of low-dose diclofenac-K.

R(+)-methanandamide-induced cyclooxygenase-2 expression in H4 human neuroglioma cells: possible involvement of membrane lipid rafts.

Cannabinoids induce the expression of the cyclooxygenase-2 (COX-2) isoenzyme in H4 human neuroglioma cells via a pathway independent of cannabinoid- or vanilloid receptor activation. The underlying mechanism was recently shown to involve increased synthesis of ceramide, which in turn leads to activation of p38 and p42/44 mitogen-activated protein kinases (MAPKs). The present study investigates a possible contribution of membrane lipid rafts to cannabinoid-induced COX-2 expression.

A liquid chromatography-mass spectrometry method for the quantification of both etoricoxib and valdecoxib in human plasma.

A practicable and selective liquid chromatography-mass spectrometry assay for the determination of two cyclooxygenase-2 inhibitors, etoricoxib and valdecoxib, in human plasma is presented. The analytical technique is based on reversed-phase high-performance liquid chromatography (HPLC) coupled to atmospheric pressure chemical ionisation (APCI) mass spectrometry (Finnigan Mat LCQ ion trap). Mass analysis was performed in the positive ion mode.

The impact of unlicensed and off-label drug use on adverse drug reactions in paediatric patients.

Background And Objective: Many drugs that are used to treat children are either not licensed for use in paediatric patients (unlicensed) or prescribed outside the terms of the product licence (off label). The incidence of adverse drug reactions (ADRs) associated with the use of such drugs is yet to be established. This study investigates, for the first time in a German patient population, the impact of unlicensed and off-label drug use on ADRs in paediatric patients.