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Aims: A CYP2C9-dependent endothelium-derived hyperpolarizing factor (EDHF) controls blood flow in many microvascular beds of various species by targeting vascular smooth muscle potassium channels. Since platelets express the same channels, we tested whether EDHF hyperpolarizes platelets and exerts an antithrombotic function in vivo., Methods and Results: Interaction of injected human platelets with the arteriolar wall (platelet-vessel wall interaction, PVWI) was assessed by intravital microscopy in skin muscle of awake hamsters. To understand the mechanisms of EDHF-induced platelet inhibition, we studied whether cultured human umbilical vein endothelial cells overexpressing CYP2C9-mRNA in vitro released a factor that could hyperpolarize human platelets. Under control conditions, there was no firm adhesion of platelets to the arteriolar wall, but temporary PVWI occurred. Local superfusion of the CYP2C9 inhibitor sulfaphenazole, at doses known to block EDHF-dependent dilations, significantly augmented PVWI, as did inhibition of NO synthase. Inhibition of both factors exerted additive effects on PVWI. Likewise, firm adhesion of a small fraction of platelets was observed. The prothrombotic effects of CYP2C9 inhibition in vivo were reversed by exogenous superfusion with 11,12-epoxyeicosatrienoic acids. Hyperpolarization reduced platelet adhesion to endothelial cells under static conditions in vitro and was dependent on calcium-activated potassium channels. The factor also reduced ADP-induced expression of platelet P-selectin, indicating reduction of platelet activity., Conclusion: The arteriolar endothelium in vivo continuously releases a CYP2C9-derived EDHF. This EDHF exerts its effects by hyperpolarization of platelets through activation of K(Ca) channels and reduction of platelet adhesion molecule expression, indicating that hyperpolarization reduces platelet activation. This demonstrates that EDHF is part of the antithrombotic properties of healthy endothelium in vivo.

Ionic liquid matrices are a new class of matrices used in MALDI mass spectrometry. The ablation process of several ionic liquid matrices was studied by determining the velocity distribution of ablated neutral matrix molecules. This was done by a postionization approach, where the neutrals were ionized in the ablation plume by a second laser pulse. It was found that a second, time-delayed ablation event occurs consisting completely of neutral molecules. To explain this, the reflected-shockwave model is used, which assumes that the shockwave emerging from the laser ablation is reflected at the sample holder surface. When the shockwave arrives at the sample surface it causes a second ablation.

The zebrafish MAGUK protein Nagie oko is a member of the evolutionarily conserved Crumbs protein complex and functions as a scaffolding protein involved in the stabilization of multi-protein assemblies at the tight junction. During zebrafish embryogenesis, mutations in nagie oko cause defects in both epithelial polarity and cardiac morphogenesis. We used deletion constructs of Nagie oko in functional rescue experiments to define domains essential for cell polarity, maintenance of epithelial integrity and cardiac morphogenesis. Inability of Nagie oko to interact with Crumbs proteins upon deletion of the PDZ domain recreates all aspects of the nagie oko mutant phenotype. Consistent with this observation, apical localization of Nagie oko within the myocardium and neural tube is dependent on Oko meduzy/Crumbs2a. Disruption of direct interactions with Patj or Lin-7, two other members of the Crumbs protein complex, via the bipartite L27 domains produces only partial nagie oko mutant phenotypes and does not impair correct junctional localization of the truncated Nagie oko deletion protein within myocardial cells. Similarly, loss of the evolutionarily conserved region 1 domain, which mediates binding to Par6, causes only a subset of the nagie oko mutant epithelial phenotypes. Finally, deletion of the C-terminus, including the entire guanylate kinase and the SH3 domains, renders the truncated Nagie oko protein inactive and recreates all features of the nagie oko mutant phenotype when tested in functional complementation assays. Our observations reveal a previously unknown diversity of alternative multi-protein assembly compositions of the Crumbs-Nagie-oko and Par6-aPKC protein complexes that are highly dependent on the developmental context.

Endothelial cell survival is indispensable to maintain endothelial integrity and initiate new vessel formation. We investigated the role of SHP-2 in endothelial cell survival and angiogenesis in vitro as well as in vivo. SHP-2 function in cultured human umbilical vein and human dermal microvascular endothelial cells was inhibited by either silencing the protein expression with antisense-oligodesoxynucleotides or treatment with a pharmacological inhibitor (PtpI IV). SHP-2 inhibition impaired capillary-like structure formation (p < 0.01; n = 8) in vitro as well as new vessel growth ex vivo(p < 0.05; n = 10) and in vivo in the chicken chorioallantoic membrane (p < 0.01, n = 4). Additionally, SHP-2 knock-down abrogated fibroblast growth factor 2 (FGF-2)-dependent endothelial proliferation measured by MTT reduction (p < 0.01; n = 12). The inhibitory effect of SHP-2 knock-down on vessel growth was mediated by increased endothelial apoptosis (annexin V staining, p < 0.05, n = 9), which was associated with reduced FGF-2-induced phosphorylation of phosphatidylinositol 3-kinase (PI3-K), Akt and extracellular regulated kinase 1/2 (ERK1/2) and involved diminished ERK1/2 phosphorylation after PI3-K inhibition (n = 3). These results suggest that SHP-2 regulates endothelial cell survival through PI3-K-Akt and mitogen-activated protein kinase pathways thereby strongly affecting new vessel formation. Thus, SHP-2 exhibits a pivotal role in angiogenesis and may represent an interesting target for therapeutic approaches controlling vessel growth., (Copyright (c) 2007 S. Karger AG, Basel.)

Non-steroidal anti-inflammatory drugs (NSAID) target the enzyme cyclooxygenase (COX) thus affording relieve from pain, inflammation or fever. As COX-dependently formed prostanoids not only mediate signals involved in inflammation and pain, but also regulate important physiological cardiovascular functions, some NSAID have recently been reported to be associated with arterial thrombosis or hypertension. This is in contrast to the well-known antiplatelet effects of low-dose aspirin, but in coherence with the specific effects of some NSAID on prostanoid formation in the vasculature. A correlation between the intake of selective inhibitors of the cyclooxygenase 2 (COX-2) isoform and atherothrombotic events has recently been established. Large retrospective analyses of clinical data have repeatedly shown this effect and in some cases have also observed potential hazards for other, rather non-selective NSAID. This review evaluates potential prothrombotic effects of NSAID in vascular ischemic disease in comparison to low-dose aspirin and selective COX-2 inhibitors and discusses pathophysiological backgrounds for such observations.

Color variants of green fluorescent protein (GFP) are increasingly used for multicolor imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP). Here we show that experimental settings commonly used in these imaging experiments may induce an as yet uncharacterized reversible photobleaching of fluorescent proteins, which is more pronounced at acidic pH. Whereas the reversible photobleaching spectrum of eCFP corresponds to its absorption spectrum, reversible photobleaching spectra of yellow variants resemble absorption spectra of their protonated states. Fluorescence intensities recover spontaneously with time constants of 25-58 s. The recovery of eCFP can be further accelerated by illumination. The resulting steady-state fluorescence reflects a variable equilibrium between reversible photobleaching, spontaneous recovery, and light-induced recovery. These processes can cause significant artifacts in commonly applied imaging techniques, photobleach-based FRET determinations, and FRAP assays.

The vanilloid receptor-related TRP channels (TRPV1-6) mediate thermosensation, pain perception and epithelial Ca(2+) entry. As the specificity of TRPV channel heteromerization and determinants governing the assembly of TRPV subunits were largely elusive, we investigated the TRPV homo- and heteromultimerization. To analyze the assembly of TRPV subunits in living cells, we generated fluorescent fusion proteins or FLAG-tagged TRPV channel subunits. The interaction between TRPV subunits was assessed by analysis of the subcellular colocalization, fluorescence resonance energy transfer and coimmunoprecipitation. Our results demonstrate that TRPV channel subunits do not combine arbitrarily. With the exception of TRPV5 and TRPV6, TRPV channel subunits preferentially assemble into homomeric complexes. Truncation of TRPV1, expression of cytosolic termini of TRPV1 or TRPV4 and construction of chimeric TRPV channel subunits revealed that the specificity and the affinity of the subunit interaction is synergistically provided by interaction modules located in the transmembrane domains and in the cytosolic termini. The relative contribution of intramolecularly linked interaction modules presumably controls the overall affinity and the specificity of TRPV channel assembly.

The low extracellular pH of inflamed or ischemic tissues enhances painful sensations by sensitizing and activating the vanilloid receptor 1 (TRPV1). We report here that activation of TRPV1 results in a marked intracellular acidification in nociceptive dorsal root ganglion neurons and in a heterologous expression system. A characterization of the underlying mechanisms revealed a Ca(2+)-dependent intracellular acidification operating at neutral pH and an additional as yet unrecognized direct proton conductance through the poorly selective TRPV1 pore operating in acidic extracellular media. Large organic cations permeate through the activated TRPV1 pore even in the presence of physiological concentrations of Na(+), Mg(2+), and Ca(2+). The wide pore and the unexpectedly high proton permeability of TRPV1 point to a proton hopping permeation mechanism along the water-filled channel pore. In acidic media, the high relative proton permeability through TRPV1 defines a novel proton entry mechanism in nociceptive neurons.

Objectives: The study investigates the correlation between left ventricular function and QRS duration obtained by alternate right ventricular pacing sites., Background: 1. Right ventricular apical pacing is associated with alterations of left ventricular contraction sequence. 2. A stimulation producing narrow QRS complexes is supposed to provide for better left ventricular contraction patterns., Methods: Fourteen patients with third degree AV block received one ventricular pacing lead in apical position. The alternate lead was attached to that site on the septum that produced the smallest QRS complex as measured from the earliest to the last deflection in any of the orthogonal Frank leads (xyz). During atrial synchronous ventricular pacing, the AV delay was optimized individually and for each stimulation site using mitral valve doppler or impedance cardiography. By radionuclide ventriculography, the phase distribution histogram of left ventricular contraction was evaluated as area under the curve (AuC); systolic function was determined as ejection fraction (EF) and as absolute ejected counts (EC) in random order. The difference (delta) in QRS duration between apical and septal stimulation (deltaxyz) was correlated with the difference in phase distribution (deltaAuC) and ejection parameters (deltaEF, deltaEC)., Results: QRS duration was shorter with septal than with apical pacing in 9 out of 14 patients (64%); it was longer in 4 (29%), and no difference was seen in 1 patient. There was a significant positive correlation between the change in QRS duration (deltaxvz) and phase distribution (deltaAuC: r = 0.66393, p = 0.010) and a significant negative correlation to systolic function (deltaEF: r = 0.70931, p = 0.004; deltaEC: r = 0.74368, p = 0.002)., Conclusions: In atrial synchronous right ventricular pacing, if the AV delay is adapted individually, decreased QRS duration obtained by alternate pacing sites is significantly correlated with homogenization of left ventricular contraction and with increased systolic function in acute tests.

The detection of preserved glucose uptake in hypoperfused dysfunctional myocardium by fluorine-18 deoxyglucose (FDG) positron emission tomography (PET) represents the method of choice in myocardial viability diagnostics. As the technique is not available for the majority of patients due to cost and the limited capacity of the PET centres, it was the aim of the present work to develop and test FDG single-photon emission tomography (SPET) with the means of conventional nuclear medicine. The perfusion marker sestamibi (MIBI) was used together with the metabolic tracer FDG in dual-isotope acquisition. A conventional SPET camera was equipped with a 511-keV collimator and designed to operate with simultaneous four-channel acquisition. In this way, the scatter of 18F into the technetium-99m energy window could be taken into account by a novel method of scatter correction. Thirty patients with regional wall motion abnormalities at rest were investigated. The results of visual wall motion analysis by contrast cine-ventriculography in nine segments/heart were compared with the results of quantitative scintigraphy. The scintigraphic patterns of MIBI and FDG tracer accumulation were defined as normal, matched defects and perfusion-metabolism mismatches. Spatial resolution of the system was satisfactory, with a full width at half maximum (FWHM) of 15.2 mm for 18F and 14.0 mm for 99mTc, as measured by planar imaging in air at 5 cm distance from the collimator. Image quality allowed interpretation in all 30 patients. 88% of segments without relevant wall motion abnormalities presented normal scintigraphic results. Seventy-five akinetic segments showed mismatches in 27%, matched defects in 44% and normal perfusion in 29%. We conclude that FDG-MIBI dual-isotope SPET is technically feasible with the means of conventional nuclear medicine. Thus, the method is potentially available for widespread application in patient care and may represent an alternative to the 201Tl reinjection technique.

In order to examine the effect of fenofibrate on coronary narrowings, within the framework of a prospective intervention study, we treated a total of 44 hypercholesterolemic patients (who were in our clinic to undergo PTCA) with diet and fenofibrate (200-400 mg/day) over a period of 3 years. After a mean interval of 21 months, control angiographies were performed in nearly identical projections for 21 patients on clinical grounds. The minor and medium-grade narrowings of the reangiographed patients at the beginning and at the end of the intervention interval were measured by means of digital image processing and automatic contour detection. The measuring parameters were percent diameter reduction (% DR) and percent plaque area (%PA). With regard to their angiographic progression, the 21 reangiographed patients of the intervention group were compared to a comparison group consisting likewise of 21 patients of similar age and sex distribution and persistently high lipid and lipoprotein levels. During the intervention period, the reangiographed patients of the intervention group showed the following changes of the lipid and lipoprotein levels in contrast to the outset values: cholesterol -19 +/- 8%, LDL -20 +/- 14%, HDL +19 +/- 44%, triglycerides -30 +/- 31%.(ABSTRACT TRUNCATED AT 250 WORDS)

To study the effects of fenofibrate, a lipid-lowering medication, on patients with coronary artery disease, 191 minor coronary narrowings in 42 patients with coronary artery disease were analyzed by quantitative coronary angiography using computer-assisted contour detection. Computed parameters were percent diameter reduction and percent plaque area. A prospectively formed intervention group of 21 patients treated with special diet and fenofibrate (200 to 400 mg/day) was checked every 6 weeks with regard to risk factors. After a mean interval of 21 months, coronary angiography was repeated, using the same x-ray system and nearly identical projections. The intervention group was angiographically compared at follow-up with an untreated comparison group, also comprising 21 patients. Both groups had high initial serum cholesterol (mean 311 mg/dl) and low-density lipoprotein (LDL) cholesterol levels (mean 235 mg/dl). Only among the treated patients did lipid levels change significantly: cholesterol, -19%; LDL cholesterol, -20%; high-density lipoprotein cholesterol, +19%; and triglycerides, -30%. At angiographic follow-up, the changes in percent diameter reduction and percent plaque area correlated positively with the mean serum and LDL cholesterol levels of the intervention group. Significant differences were found in the change in percent plaque area between both groups. The intervention subgroup with angiographic regressions (11 patients) had significantly lower serum and LDL cholesterol levels than the intervention subgroup with angiographic progressions (10 patients). These results indicate the beneficial effect of fenofibrate on minor coronary narrowings. Because of its high reproducibility in measuring minor narrowings, quantitative coronary angiography proved to be a suitable method for angiographic follow-up.