The role of the cardiac myocyte as a mediator of paracrine signaling in the heart has remained unclear. To address this issue, we generated mice with cardiac myocyte-specific deletion of the vascular endothelial growth factor gene, thereby producing a cardiomyocyte-specific knockout of a secreted factor. The hearts of these mice had fewer coronary microvessels, thinned ventricular walls, depressed basal contractile function, induction of hypoxiaresponsive genes involved in energy metabolism, and an abnormal response to ␤-adrenergic stimulation. These findings establish the critical importance of cardiac myocyte-derived vascular endothelial growth factor in cardiac morphogenesis and determination of heart function. Further, they establish an adult murine model of hypovascular nonnecrotic cardiac contractile dysfunction.

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Three glycolytic enzymes, hexokinase, phosphoglycerate kinase, and pyruvate kinase, were fluorine labeled in the yeast Saccharomyces cerevisiae by biosynthetic incorporation of 5-fluorotryptophan. 19F NMR longitudinal relaxation time measurements on the labeled enzymes were used to assess their rotational mobility in the intact cell. Comparison with the results obtained from relaxation time measurements of the purified enzymes in vitro and from theoretical calculations showed that two of the labeled enzymes, phosphoglycerate kinase and hexokinase, were tumbling in a cytoplasm that had a viscosity approximately twice that of water. There were no detectable signals from pyruvate kinase in vivo, although it could be detected in diluted cell extracts, indicating that there was some degree of motional restriction of the enzyme in the intact cell.

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BackgroundThe BRAF inhibitor, vemurafenib, has recently been approved for the treatment of metastatic melanoma in patients harboring BRAFV600 mutations. Currently, dual BRAF and MEK inhibition are ongoing in clinical trials with the goal of overcoming the acquired resistance that has unfortunately developed in some vemurafenib patients. FDG-PET measures of metabolic activity are increasingly employed as a pharmacodynamic biomarker for guiding single-agent or combination therapies by gauging initial drug response and monitoring disease progression. However, since tumors are inherently heterogeneous, investigating the effects of BRAF and MEK inhibition on FDG uptake in a panel of different melanomas could help interpret imaging outcomes.Methods18 F-FDG uptake was measured in vitro in cells with wild-type and mutant (V600) BRAF, and in melanoma cells with an acquired resistance to vemurafenib. We treated the cells with vemurafenib alone or in combination with MEK inhibitor GDC-0973. PET imaging was used in mice to measure FDG uptake in A375 melanoma xenografts and in A375 R1, a vemurafenib-resistant derivative. Histological and biochemical studies of glucose transporters, the MAPK and glycolytic pathways were also undertaken.ResultsWe demonstrate that vemurafenib is equally effective at reducing FDG uptake in cell lines harboring either heterozygous or homozygous BRAFV600 but ineffective in cells with acquired resistance or having WT BRAF status. However, combination with GDC-0973 results in a highly significant increase of efficacy and inhibition of FDG uptake across all twenty lines. Drug-induced changes in FDG uptake were associated with altered levels of membrane GLUT-1, and cell lines harboring RAS mutations displayed enhanced FDG uptake upon exposure to vemurafenib. Interestingly, we found that vemurafenib treatment in mice bearing drug-resistant A375 xenografts also induced increased FDG tumor uptake, accompanied by increases in Hif-1α, Sp1 and Ksr protein levels. Vemurafenib and GDC-0973 combination efficacy was associated with decreased levels of hexokinase II, c-RAF, Ksr and p-MEK protein.ConclusionsWe have demonstrated that 18 F-FDG-PET imaging reflects vemurafenib and GDC-0973 action across a wide range of metastatic melanomas. A delayed post-treatment increase in tumor FDG uptake should be considered carefully as it may well be an indication of acquired drug resistance.Trial registrationClinicalTrials.gov NCT01271803

ACE inhibition after MI inhibits cardiac hypertrophy, preserves cardiac function, and attenuates changes in myocardial gene expression. Gene expression profiling reveals, however, that some elements of the pathophysiology may be unaffected by the treatment and be targets for new therapies.

ASummary: Contrast-enhanced magnetic resonance imaging was used to produce high-resolution activation maps reflecting local changes in cerebral blood volume after a simple sensory stimulus, Activation of the forelimb region of the somatosen sory cortex was performed in a-chloralose-anaesthetized rats with an electrical stimulus (5 V, 3 Hz) delivered through needle electrodes placed subcutaneously on the left forelimb, A gra dient echo magnetic resonance imaging sequence, sensitive to Recovery of brain function and reorganization of sen sory and motor maps after deprivation of sensory input or local damage is an active area of neuroscience re search. Whereas it is recognized that the adult brain is able to undergo considerable plastic changes to affect restitution of function, much of the underlying neuro physiologic mechanisms remain unknown. The cortex of the rat provides a valuable model with which to study recovery of function. The cytoarchitecture is accurately documented, and its functional physiologic features are well characterized. Studies on this subject have relied on 1178changes in the relative amount of deoxyhemoglobin within the cerebral vasculature, produced a 4,05% ± 1.69% increase in signal intensity, This effect was enhanced with an injection of an intravascular iron oxide contrast agent (Combidex, Ad vanced Magnetics), resulting in a 9, II % ± 1.52% decrease in signal intensity. Key Words: Functional magnetic resonance imaging-Somatosensory activation-Iron oxide contrast agent-Cerebral blood volume-Combidex.techniques that are either invasive (eg, involve electrode placement) or lack the spatial resolution necessary when working with laboratory animals. Recent developments in magnetic resonance (MR) imaging technology have allowed the spatial distribution of functional neuronal activity to be mapped noninvasively. Whereas most of these studies have been confined to human cognition (Kwong et aI., 1992; Bandettini et aI., 1992;Moseley and Glover, 1995), functional magnetic resonance imaging (fMRI) provides an attractive alternative for functional mapping in the laboratory animal.During somatosensory stimulation, local alteration in neuronal activity induces local changes in metabolism and CBF and volume (Fox and Raichle, 1986). This re sults in a change in the local concentration of deoxyhe moglobin within the cerebral vasculature (Ogawa et aI., 1990; Turner et aI., 1991) and, because of the paramag netic properties of deoxyhemoglobin, causes a change in local T2 and T2* nuclear magnetic resonance relaxation time (Thulborn et aI., 1982). This effect has been termed blood oxygenation level-dependent (BOLD) image con trast. Several studies have demonstrated the feasibility of performing fMRI in rats exploiting the BOLD effect (Kerskens et aI

Abstract. Molecular imaging techniques for protein therapeutics rely on reporter labels, especially radionuclides or sometimes near-infrared fluorescent moieties, which must be introduced with minimal perturbation of the protein's function in vivo and are detected non-invasively during whole-body imaging. PET is the most sensitive whole-body imaging technique available, making it possible to perform biodistribution studies in humans with as little as 1 mg of injected antibody carrying 1 mCi (37 MBq) of zirconium-89 radiolabel. Different labeling chemistries facilitate a variety of optical and radionuclide methods that offer complementary information from microscopy and autoradiography and offer some trade-offs in whole-body imaging between cost and logistic difficulty and image quality and sensitivity (how much protein needs to be injected). Interpretation of tissue uptake requires consideration of label that has been catabolized and possibly residualized. Image contrast depends as much on background signal as it does on tissue uptake, and so the choice of injected dose and scan timing guides the selection of a suitable label and helps to optimize image quality. Although only recently developed, zirconium-89 PET techniques allow for the most quantitative tomographic imaging at millimeter resolution in small animals and they translate very well into clinical use as exemplified by studies of radiolabeled antibodies, including trastuzumab in breast cancer patients, in The Netherlands.