Heide L. Kirschenlohr

Proton NMR analysis of plasma is a weak predictor of coronary artery disease

Multivariate analysis of 1H-NMR spectra of blood sera was reported previously to predict angiographically defined advanced coronary artery disease (CAD) with >90% accuracy and specificity. The analysis depended mainly on the major lipid regions of the spectra, but many variables, including gender and drug treatment, affect lipid composition and are potential confounders. We have determined the predictive power of the same methodology for angiographically defined CAD using plasma samples from groups of male patients, classified by statin treatment, who had normal coronary arteries (NCAs) or CAD. Predictions for NCA and CAD groups were only 80.3% correct for patients not treated with statins and 61.3% for treated patients, compared with random correct predictions of 50%. A confidence limit of >99% was achieved for 36.2% of predictions for untreated groups and 6.2% for treated groups. Detection of CAD by 1H-NMR with >99% confidence was therefore very weak compared with angiography.

Tamoxifen Effects on Endothelial Function and Cardiovascular Risk Factors in Men With Advanced Atherosclerosis

BackgroundTamoxifen and its analogues act as selective estrogen receptor modulators (SERMs) in women, with estrogen-like activities on some plasma cardiovascular risk factors (eg, lipoproteins). Effects of SERMs on men with coronary artery disease (CAD) have not been reported. Methods and ResultsThirty-one men with angiographically proven CAD were recruited; 16 were treated with tamoxifen (40 mg/d) for 56 days, and 15 were untreated. All the CAD patients were medicated with aspirin and an HMG-CoA reductase inhibitor for ≥6 weeks before entering the study. Ten men with angina-like symptoms but normal coronary arteries by angiography (NCA group) were also treated with tamoxifen. Blood samples were collected at days −7, 0,7, 14, 21, 28, and 56 of treatment. Endothelium-dependent flow-mediated dilatation (ED-FMD) of the brachial artery was measured by high-resolution ultrasound at 5 visits. Tamoxifen caused an increase in %ED-FMD maximal at 28 days in the CAD group (2.1±0.3% to 7.5±0.7%;P <0.0001) and the NCA group (3.8±0.4% to 7.9±1.0%;P <0.0001), with no significant change in the untreated group. Tamoxifen also caused decreases in several plasma cardiovascular risk factors, including total cholesterol, triglycerides, lipoprotein(a), and fibrinogen. Except for the triglyceride response, these effects were similar to those reported for postmenopausal women treated with tamoxifen. ConclusionsTamoxifen substantially increased ED-FMD in men with CAD who were taking conventional medication. Together with the effects on risk factors, the data strongly support clinical evaluation of SERMs for the treatment of men with CAD.

Ca2+ buffering in the heart: Ca2+ binding to and activation of cardiac myofibrils.

The measurement of cardiac Ca(2+) transients using spectroscopic Ca(2+) indicators is significantly affected by the buffering properties of the indicators. The aim of the present study was to construct a model of cardiac Ca(2+) buffering that satisfied the kinetic constraints imposed by the maximum attainable rates of cardiac contraction and relaxation on the Ca(2+) dissociation rate constants and which would account for the observed effects of (19)F-NMR indicators on the cardiac Ca(2+) transient in the Langendorff-perfused ferret heart. It is generally assumed that the Ca(2+) dependency of myofibril activation in cardiac myocytes is mediated by a single Ca(2+)-binding site on troponin C. A model based on 1:1 Ca(2+) binding to the myofilaments, however, was unable to reproduce our experimental data, but a model in which we assumed ATP-dependent co-operative Ca(2+) binding to the myofilaments was able to reproduce these data. This model was used to calculate the concentration and dissociation constant of the ATP-independent myofilament Ca(2+) binding, giving 58 and 2.0 microM respectively. In addition to reproducing our experimental data on the concentration of free Ca(2+) ions in the cytoplasm ([Ca(2+)](i)), the resulting Ca(2+) and ATP affinities given by fitting of the model also provided good predictions of the Ca(2+) dependence of the myofibrillar ATPase activity measured under in vitro conditions. Solutions to the model also indicate that the Ca(2+) mobilized during each beat remains unchanged in the presence of the additional buffering load from Ca(2+) indicators. The new model was used to estimate the extent of perturbation of the Ca(2+) transient caused by different concentrations of indicators. As little as 10 microM of a Ca(2+) indicator with a dissociation constant of 200 nM will cause a 20% reduction in peak-systolic [Ca(2+)](i) and 30 microM will cause approx. 50% reduction in the peak-systolic [Ca(2+)](i) in a heart paced at 1.0 Hz.

Cultures of Proliferating Vascular Smooth Muscle Cells from Adult Human Aorta

Abnormal proliferation of human vascular smooth muscle cells (hVSMCs) is a central event in the development of atherosclerosis (1-3) As a result, there is considerable interest in the establishment of hVSMC cultures as a mode1 of this disease process However, it has been noted in the past (4,5) that hVSMCs, especially when cultured by the enzyme-dispersal technique (hVSMC,(ED)), grow poorly in culture compared to VSMCs from other species (e.g., rat) This has limited their use for cell-culture studies. We have recently reported that the reduced proliferative capacity of hVSMC(ED) from adult aorta can be attributed to the endogenous production of active TGF-β(6,7). We (6-8) and others (9-11) have shown that TGF-β; is a potent inhibtior of smooth muscle cell proliferation. Moreover, recent studies in animal models of atherosclerosis (12) have suggested that TGF-P plays a pivotal role in regulation of vessel wall architecture (13). We have also shown that hVSMCs derived by the alternative method of explanting (hVSMC(EX)) have a greater proliferative capacity than the hVSMC(ED) (14) In accordance with our hypotheses, the cells grown from explanted tissue did not produce TGF-β; (14).

Gene Expression Profiles in White Blood Cells of Volunteers Exposed to a 50 Hz Electromagnetic Field

Consistent and independently replicated laboratory evidence to support a causative relationship between environmental exposure to extremely low-frequency electromagnetic fields (EMFs) at power line frequencies and the associated increase in risk of childhood leukemia has not been obtained. In particular, although gene expression responses have been reported in a wide variety of cells, none has emerged as robust, widely replicated effects. DNA microarrays facilitate comprehensive searches for changes in gene expression without a requirement to select candidate responsive genes. To determine if gene expression changes occur in white blood cells of volunteers exposed to an ELF-EMF, each of 17 pairs of male volunteers age 20–30 was subjected either to a 50 Hz EMF exposure of 62.0 ± 7.1 μT for 2 h or to a sham exposure (0.21 ± 0.05 μT) at the same time (11:00 a.m. to 13:00 p.m.). The alternative regime for each volunteer was repeated on the following day and the two-day sequence was repeated 6 days later, with the exception that a null exposure (0.085 ± 0.01 μT) replaced the sham exposure. Five blood samples (10 ml) were collected at 2 h intervals from 9:00 to 17:00 with five additional samples during the exposure and sham or null exposure periods on each study day. RNA samples were pooled for the same time on each study day for the group of 17 volunteers that were subjected to the ELF-EMF exposure/sham or null exposure sequence and were analyzed on Illumina microarrays. Time courses for 16 mammalian genes previously reported to be responsive to ELF-EMF exposure, including immediate early genes, stress response, cell proliferation and apoptotic genes were examined in detail. No genes or gene sets showed consistent response profiles to repeated ELF-EMF exposures. A stress response was detected as a transient increase in plasma cortisol at the onset of either exposure or sham exposure on the first study day. The cortisol response diminished progressively on subsequent exposures or sham exposures, and was attributable to mild stress associated with the experimental protocol.

Investigation of transcriptional responses of juvenile mouse bone marrow to power frequency magnetic fields.

To seek alterations in gene transcription in bone marrow cells following in vivo exposure of juvenile mice to power frequency magnetic fields, young (21-24-day old) C57BL/6 mice were exposed to a 100μT 50Hz magnetic field for 2h. Transcription was analysed by three methods, High Coverage Expression Profiling (HiCEP), Illumina microarrays and quantitative real-time polymerase chain reaction (QRT-PCR). A pilot HiCEP experiment with 6 exposed (E) and 6 non-exposed (NE) mice identified four candidate responsive transcripts (two unknown transcripts (AK152075 and F10-NED), phosphatidylinositol binding clathrin assembly protein (Picalm) and exportin 7 (Xpo7)). A larger experiment compared 19 E and 15 NE mice using two independent QRT-PCR assays and repeated microarray assays. No significant field-dependent changes were seen, although Picalm showed a trend to significance in one QRT-PCR assay (E/NE=0.91; P=0.06). However, the study was underpowered to detect an effect of this magnitude (52% power at P=0.05). These data indicate the current experimental constraints in detecting small changes in transcription that may occur in response to magnetic fields. These constraints result from technical limitations in the accuracy of assays and biological variation, which together were sufficient to account statistically for the number of differentially expressed transcripts identified in the pilot experiment.

A new 19F NMR indicator for intracellular sodium

The effect of increased benzo substitution on the cation-binding strength of diaza-18-crown-6 has been investigated. The planarity constraint imposed by increased substitution reduces the effective cavity size thus giving rise to an increased selectivity for sodium over potassium, magnesium and calcium that is sufficient to provide a class of indicators suitable for measuring intracellular free sodium. The cation binding parameters and the associated spectral changes of a selection of compounds with varied aryl fluorination patterns have shown that there is very little electronic interaction between the aromatic rings and the bound cation through the heteroatoms of the crown ether. Only small adjustments to the cation affinity can therefore be achieved via alteration of the aryl substitution patterns. Aryl substitution adjacent to the hetero atoms gives a reduction in the cation affinity of the tribenzo-crown ether, similar to that found on fluorination of the calcium chelator BAPTA ortho to the oxyethylene bridge. These observations have led to the preparation of a new indicator for intracellular sodium that we have called FCrown-1. The new indicator has a dissociation constant for sodium of 11 mmol dm–3 and a selectivity for sodium over potassium of 173-fold. The sodium concentration is reported by the chemical shift (fast exchange) of one of the two types of fluorine present with a maximum sodium-induced shift of 4.6 ppm downfield. The resonance arising from the other fluorine substituents is insensitive to sodium and acts as an internal chemical shift reference at 8.8 to 13.4 ppm upfield from the reporting signal.

Analysis of Heterogeneity in T2-Weighted MR Images Can Differentiate Pseudoprogression from Progression in Glioblastoma

Funded by Medical Research Council/ Royal College of Radiologists (UK) Clinical Research Fellowship (G1000265); Cancer Research UK Clinical Research Fellowship; Addenbrookes Charitable Trust Award to TCB. Cancer Research UK Programme grant (C197/ A3514) to KMB.