Local delivery of the KCa3.1 blocker, TRAM-34, prevents acute angioplasty-induced coronary smooth muscle phenotypic modulation and limits stenosis

Objective-We previously demonstrated that upregulation of intermediate-conductance Ca2+ -activated K+ channels (KCa 3.1) is necessary for mitogen-induced phenotypic modulation in isolated porcine coronary smooth muscle cells (SMCs). The objective of the present study was to determine the role of KCa3.1 in the regulation of coronary SMC phenotypic modulation in vivo using a swine model of postangioplasty restenosis. Methods and Results-Balloon angioplasty was performed on coronary arteries of swine using either noncoated or balloons coated with the specific KCa3.1 blocker TRAM-34. Expression of KCa3.1, c-jun, c-fos, repressor element-1 silencing transcription factor (REST), smooth muscle myosin heavy chain (SMMHC), and myocardin was measured using qRT-PCR in isolated medial cells 2 hours and 2 days postangioplasty. KCa3.1, c-jun, and c-fos mRNA levels were increased 2 hours postangioplasty, whereas REST expression decreased. SMMHC expression was unchanged at 2 hours, but decreased 2 days postangioplasty. Use of TRAM-34 coated balloons prevented KCa3.1 upregulation and REST downregulation at 2 hours, SMMHC and myocardin downregulation at 2 days, and attenuated subsequent restenosis 14 and 28 days postangioplasty. Immunohistochemical analysis demonstrated corresponding changes at the protein level. Conclusion-Blockade of KCa3.1 by delivery of TRAM-34 via balloon catheter prevented smooth muscle phenotypic modulation and limited subsequent restenosis. © 2008 American Heart Association, Inc.

Stress-induced platelet formation in silicon:a molecular dynamics study

The effect of stress on vacancy cluster configurations in silicon is examined using molecular dynamics. At zero pressure, the shape and stability of the vacancy clusters agrees with previous atomistic results. When stress is applied the orientation of small planar clusters changes to reduce the strain energy. The preferred orientation for the vacancy clusters under stress agrees with the experimentally observed orientations of hydrogen platelets in the high stress regions of hydrogen implanted silicon. These results suggest a theory for hydrogen platelet formation. © 2005 The American Physical Society.

Investigations of the site and mechanisms of drug-induced reductions in the reabsorption of divalent cations by the kidney

Disturbances in electrolyte homeostasis are a frequent adverse side-effect of the administration of aminoglycoside antibiotics such as gentamicin, and the antineoplastic agent cis-platinum. The aims of this work were to further elucidate the site(s) and mechanism(s) by which these drugs may produce disturbances in the renal reabsorption of calcium and magnesium. These investigations were undertaken using a range of in vivo and in vitro techniques and models. Initially, a series of in vivo studies was conducted to delineate aspects of the acute and chronic effects of both drugs on renal electrolyte handling and to select and evaluate an appropriate animal model: subsequent investigations were focused on gentamicin. In a study of the acute and chronic effects of cis-platinum administration, there were pronounced acute changes in a variety of indices of nephrotoxic injury, including electrolyte excretion. Most effects resolved but there were chronic increases in the urinary excretion of calcium and magnesium. The renal response of three strains of rat (Fischer 344, Sprague-Dawley (SD), and Wistar) to a ranges of doses of gentamicin was also investigated. Drug administration produced substantially different responses between strains, in particular marked differences in calcium and magnesium excretion. The results suggested that the SD rat was an appropriately sensitive strain for use in further investigations. Acute infusion of gentamicin in the anaesthetised SD rat produced rapid, substantial increases in the fractional excretion of calcium and magnesium, while sodium and potassium output were unaffected, confirming previous results of similar experiments using F344 rats. Studies using lithium clearance measurements in the anaesthetised SD rat were undertaken to investigate the effects of gentamicin on proximal tubular calcium reabsorption. Lithium clearance was unaffected by acute gentamicin infusion, suggesting that the site of acute gentamicin-induced hypercalciuria may not be located in the proximal tubule. Inhibition of Ca2+ ATPase activity was investigated as a potential mechanism by which calcium reabsorption could be affected after aminoglycoside administration. In vitro, both Ca2+ ATPase and Na+/K+ ATPase activity could be similarly inhibited by the presence of aminoglycosides, in a dose-related manner. Whilst inhibition of Na+/K+ ATPase could be demonstrated biochemically after in vivo administration of gentamicin, there were no concurrent effects on Ca2+ ATPase activity, suggesting that inhibition of Ca2+ ATPase activity is unlikely to be a primary mechanism of aminoglycoside-induced reductions of calcium reabsorption. Histochemical studies could not discern inhibition of either Na+/K+ ATPase or Ca2+ ATPase activity after in vivo administration of gentamicin. Selection of renal cell lines for further investigative in vitro studies on the mechanisms of altered cation reabsorption was considered using MTT (3-(4,5,-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Neutral Red cytotoxicity assays. The ability of LLC-PK1 and LLC-RK1 cell lines to correctly rank a series of nephrotoxic compounds with their known nephrotoxic potency in vivo was studied. Using these cell lines grown on semi-permeable inserts, alterations in the paracellular transport of 45Ca was investigated as a possible mechanism by which gentamicin could alter calcium reabsorption in vivo. Short term exposure (I h) of LLC-RK1 cells to gentamicin, via both cell surfaces, resulted in a reduction in paracellular permeability to both transepithelial 3H-mannitol and 45Ca fluxes. When LLC-RK1 cells were exposed via the apical surface only, similar dose-related reductions were seen to those observed when cells were exposed to the drug from both sides. Short-term basal exposure to gentamicin appeared to contribute less to the observed reductions in 3H-mannitol and 45Ca fluxes. Experiments investigating transepithelial movement of 45Ca and 3H-mannitol on LLC-PK1 cells after acute gentamicin exposure were inconclusive. Longer exposure (48 h) to gentamicin caused an increase in the permeability of the monolayer and a consequent increase in transepithelial 45Ca flux in the LLC-RK1 cell line; increases in permeability of LLC-PK1 cells to 45Ca and 3H-mannitol were not apparent under the same conditions. The site and mechanism at which gentamicin, in particular, alters calcium reabsorption cannot be definitively described from these studies. However, indirect evidence from lithium clearance studies suggests that the site of the lesion is unlikely to be located in the proximal tubule. The mechanism by which gentamicin exposure alters calcium reabsorption may be by reducing paracellular permeability to calcium rather than by altering active calcium transport processes.

Synthetic ceramides inhibit CD36 expression in U937 cells through a reduction in cytosolic peroxide

Ceramide (a sphingolipid) and reactive oxygen species (ROS) are each partly responsible for the intracellular signal transduction of a variety of physiological, pharmacological or environmental agents. It has been reported that synthesis of ceramide and ROS are intimately linked, and show reciprocal regulation. The levels of ceramide are reported to be elevated in atherosclerotic plaques providing circumstantial evidence for a pro-atherogenic role for ceramide. Indeed, LDL may be important sources of ceramide from sphingomyelin, where it promotes LDL aggregation. Using synthetic, short chain ceramides to mimic the cellular responses to fluctuations in natural endogenous ceramides, we have investigated ceramide effects on both intracellular redox state (as glutathione and ROS) and redox-sensitive gene expression, specifically the scavenger receptor CD36 (using RT-PCR and flow cytometry), in U937 monocytes and macrophages. We describe that the principal redox altering properties of ceramide are to lower cytosolic peroxide and to increase mitochondrial ROS formation, where growth arrest of U937 monocytes is also observed. In addition, cellular glutathione was depleted, which was independent of an increase in glutathione peroxidase activity. Examination of the effects of ceramide on stress induced CD36 expression in macrophages, revealed a dose dependent reduction in CD36 mRNA and protein levels, which was mimicked by N-acetyl cysteine. Taken together, these data suggest that ceramides differentially affect ROS within different cellular compartments, and that loss of cytosolic peroxide inhibits expression of the redox sensitive gene, CD36. This may attenuate both the uptake of oxidised LDL and the interaction of HDL with macrophages. The resulting sequelae in vivo remain to be determined.

RGD-independent cell adhesion via a tissue transglutaminase-fibronectin matrix promotes fibronectin fibril deposition and requires syndecan-4/2 and α5β1 integrin co-signaling

Fibronectin (FN) deposition mediated by fibroblasts is an important process in matrix remodeling and wound healing. By monitoring the deposition of soluble biotinylated FN, we show that the stress-induced TG-FN matrix, a matrix complex of tissue transglutaminase (TG2) with its high affinity binding partner FN, can increase both exogenous and cellular FN deposition and also restore it when cell adhesion is interrupted via the presence of RGD-containing peptides. This mechanism does not require the transamidase activity of TG2 but is activated through an RGD-independent adhesion process requiring a heterocomplex of TG2 and FN and is mediated by a syndecan-4 and ß1 integrin co-signaling pathway. By using a5 null cells, ß1 integrin functional blocking antibody, and a a5ß1 integrin targeting peptide A5-1, we demonstrate that the a5 and ß1 integrins are essential for TG-FN to compensate RGD-induced loss of cell adhesion and FN deposition. The importance of syndecan-2 in this process was shown using targeting siRNAs, which abolished the compensation effect of TG-FN on the RGD-induced loss of cell adhesion, resulting in disruption of actin skeleton formation and FN deposition. Unlike syndecan-4, syndecan-2 does not interact directly with TG2 but acts as a downstream effector in regulating actin cytoskeleton organization through the ROCK pathway. We demonstrate that PKCa is likely to be the important link between syndecan-4 and syndecan-2 signaling and that TG2 is the functional component of the TG-FN heterocomplex in mediating cell adhesion via its direct interaction with heparan sulfate chains.

The importance of the tissue transglutaminase-fibronectin heterocomplex in the RGD-independent cell adhesion and fibronectin matrix deposition

Tissue transglutaminase (TG2) has been reported as a wound response protein. Once over-expressed by cells under stress such as during wound healing or following tissue damage, TG2 can be secreted and deposited into extracellular matrix, where it forms a heterocomplex (TG-FN) with the abundant matrix protein fibronectin (FN). A further cellular response elicited after tissue damage is that of matrix remodelling leading to the release of the Arg-Gly-Asp (RGD) containing matrix fragments by matrix matelloproteinases (MMPs). These peptides are able to block the interaction between integrin cell surface receptors and ECM proteins, leading to the loss of cell adhesion and ultimately Anoikis. This study provides a mechanism for TG2, as a stress-induced matrix protein, in protecting the cells from the RGD-dependent loss of cell adhesion and rescuing the cells from Anoikis. Mouse fibroblasts were used as a major model for this study, including different types of cell surface receptor knockout mouse embryonic fibroblasts (MEFs) (such as syndecan-4, a5, ß1 or ß3 integrins). In addition specific syndecan-2 targetting siRNAs, ß1 integrin and a4ß1 integrin functional blocking antibodies, and a specific targeting peptide against a5ß1 integrin A5-1 were used to investigate the involvement of these receptors in the RGD-independent cell adhesion on TG-FN. Crucial for TG-FN to compensate the RGD-independent cell adhesion and actin cytoskeleton formation is the direct interaction between the heparan sulfate chains of syndecan-4 and TG2, which elicits the inside-out signalling of a5ß1 integrin and the intracellular activation of syndecan-2 by protein kinase C a (PKCa). By using specific inhibitors, a cell-permeable inhibiting peptide and the detection of the phosphorylation sites for protein kinases and/or the translocation of PKCa via Western blotting, the activation of PKCa, focal adhesion kinase (FAK), ERK1/2 and Rho kinase (ROCK) were confirmed as downstream signalling molecules. Importantly, this study also investigated the influence of TG-FN on matrix turnover and demonstrated that TG-FN can restore the RGD-independent FN deposition process via an a5ß1 integrin and syndecan-4/2 co-signalling pathway linked by PKCa in a transamidating-independent manner. These data provide a novel function for TG2 in wound healing and matrix turnover which is a key event in a number of both physiological and pathological processes.

Fatigue in rubber vulcanisates

A study has been made of the effect of single extensions and continuous fatigue on the structures of various natural rubber networks. The change in network structure of a conventional vulcanisate on a single extension manifests itself as permanent set. The change in network structure has been assessed by the use of the chemical probes, propan-2-thiol/piperidine, hexane-thiol/piperidine and triphenyl phosphine, which determine the polysulphide and disulphide crosslink densities and main chain modification respectively. The permanent set induced on a single extension of a conventional sulphur vulcanisate has been shown to result from the destruction and reformation of polysulphide crosslinks. The magnitude of the effect was dependent upon the degree of extension and showed a maximum at extensions corresponding to the onset of stress-induced crystallisation. The incorporation of a reinforcing filler, HAF-carbon black, magnified the effect. Vulcanisates that possessed only mono and disulphide crosslinks did not show any significant permanent set. The continuous changes in network structure during fatigue have also been determined, and the effects of carbon black and antioxidants on these changes and the fatigue life have been assessed. During fatigue the overall crosslink density increased slightly, which resulted from the destruction of polysulphide crosslinks. and their replacement by principally disulphide crosslinks. Antioxidants reduced the rate of destruction of polysulphide crosslinks and increased the fatigue life of the rubber network. The fatigue life of the network also depended upon the concentration of free chain ends. These chain ends were incorporated into the network by masticating rubber under nitrogen in the presence of bis (diisopropyl)thiophosphoryl disulphide, which improved the fatigue resistance by up to 9%.

Transverse load and orientation measurement with multicore fiber Bragg gratings

We demonstrate the sensitivity of Bragg gratings in a multicore fiber to transverse load. The Bragg peaks are split because of stress-induced birefringence, the magnitude of which depends upon the load and grating position relative to the load axis. Experiments show that a set of gratings in a four-core fiber can measure a load axis angle to ±5° and a load magnitude to ±15 N m-1 up to 2500 N m-1. We consider alternative designs of multicore fiber for optimal load sensing and compare experimental and modeled data. © 2005 Optical Society of America.

Microstructure-based inherent anisotropy of asphalt mixtures

Asphalt mixtures have been demonstrated to be anisotropic materials in both laboratory and field tests. The anisotropy of asphalt mixtures consists of inherent anisotropy and stress-induced anisotropy. In previous work, the inherent anisotropy of asphalt mixtures was quantified by using only the inclination angles of the coarse aggregate particles in the asphalt mixtures. However, the inclination of fine aggregates also has a contribution to the inherent anisotropy. Moreover, the contribution to the inherent anisotropy of each aggregate may not be the same as in the previous work but will depend on the size, orientation, and sphericity of the aggregate particle. This paper quantifies the internal microstructure of the aggregates in asphalt mixtures by using an aggregate-related geometric parameter, the vector magnitude. The original formulation of the vector magnitude, which addresses only the orientation of coarse aggregates, is modified to account for not only the coarse aggregate orientation, but also the size, orientation, and sphericity of coarse and fine aggregates. This formulation is applied to cylindrical lab-mixed lab-compacted asphalt mixture specimens varying in asphalt binder type, air void content, and aging period. The vertical modulus and the horizontal modulus are also measured by using nondestructive tests. A relationship between the modified vector magnitude and the modulus ratio of the vertical modulus to the horizontal modulus is developed to quantify the influence of the inherent microstructure of the aggregates on the anisotropy of the mixtures. The modulus ratio is found to depend solely on the aggregate characteristics including the inclination angle, size, and sphericity, and it is independent of the asphalt binder type, air void content, and aging period. The inclination angle, itself, proves to be insufficient to quantify the inherent anisotropy of the asphalt mixtures. © 2011 American Society of Civil Engineers.

Detemplation of soft mesoporous silica nanoparticles with structural preservation

A mild protocol that allows the template removal of soft un-aged silica nanoparticles was investigated. After oxidizing the organic template by Fenton chemistry, a good structural preservation is only achieved when the material is equilibrated and dried in a low-surface tension solvent. This avoids excessive capillary stress induced by the high surface tension of water, a major component in the Fenton reaction medium. The Fenton reaction should be carried out under mild conditions as well; otherwise the sample deteriorates by extensive hydrolysis, and capillary stress, and the structural ordering diminishes severely. We propose employing 10 ppm Fe concentration at 70 °C for 24 h for the cetyltrimethylammonium bromide template. The proposed protocol involves 2 steps resulting in an overall significantly higher pore volume attributed to the wider pores and limited particle agglomeration, while the calcined counterpart evidences aggregation and loss of the hexagonal ordering. n-BuOH exchange is unnecessary when the mesophase is stabilized by ageing, as the structure resists the water capillary stress. © The Royal Society of Chemistry 2013.