Vascular endothelial growth factor KDR receptor signaling potentiates tumor necrosis factor-induced tissue factor expression in endothelial cells

Vascular endothelial growth factor (VEGF) and tumor necrosis factor-alpha (TNF-alpha) have been shown to synergistically increase tissue factor (TF) expression in endothelial cells; however, the role of the VEGF receptors (KDR, Flt-1, and neuropilin) in this process is unclear. Here we report that VEGF binding to the KDR receptor is necessary and sufficient for the potentiation of TNF-induced TF expression in human umbilical vein endothelial cells. TF expression was evaluated by Western blot analysis and fluorescence-activated cell sorting. In the absence of TNF-alpha, wild-type VEGF- or KDR receptor-selective variants induced an approximate 7-fold increase in total TF expression. Treatment with TNF alone produced an approximate 110-fold increase in total TF expression, whereas coincubation of TNF-alpha with wild-type VEGF- or KDR-selective variants resulted in an approximate 250-fold increase in TF expression. VEGF lacking the heparin binding domain was also able to potentiate TF expression, indicating that heparin-sulfate proteoglycan or neuropilin binding is not required for TF up-regulation. Neither placental growth factor nor an Flt-1-selective variant was capable of inducing TF expression in the presence or absence of TNF. Inhibition of protein-tyrosine kinase or protein kinase C activity significantly blocked the TNF/VEGF potentiation of TF up-regulation, whereas phorbol 12-myristate 13-acetate, a protein kinase C activator, increased TF expression. These data demonstrate that KDR receptor signaling governs both VEGF-induced TF expression and the potentiation of TNF-induced up-regulation of TF.     

Diffraction-Enhanced Computed Tomographic Imaging of Growing Piglet Joints by Using a Synchrotron Light Source

The objective of this project was to develop and test a new technology for imaging growing joints by means of diffraction-enhanced imaging (DEI) combined with CT and using a synchrotron radiation source. DEI–CT images of an explanted 4-wk-old piglet stifle joint were acquired by using a 40-keV beam. The series of scanned slices was later ‘stitched’ together, forming a 3D dataset. High-resolution DEI-CT images demonstrated fine detail within all joint structures and tissues. Striking detail of vasculature traversing between bone and cartilage, a characteristic of growing but not mature joints, was demonstrated. This report documents for the first time that DEI combined with CT and a synchrotron radiation source can generate more detailed images of intact, growing joints than can currently available conventional imaging modalities.Abbreviations: DEI, diffraction-enhanced imagingDiffraction-enhanced imaging (DEI) is a biomedical imaging technique that, compared with conventional radiography, generates very detailed images with more edge contrast but deposits a lower radiation dose to the object. DEI generates enhanced contrast both from absorption, the process involved in conventional radiography, and from of X-ray refraction, a process that harnesses photons that otherwise typically are imperceptibly diffracted.⁴ The DEI technique collects information from X-rays that are refracted as they pass through tissues that have different refractive indices as it almost completely removes diffracted X-rays. In comparison, conventional radiography produces images from X-rays that are attenuated by the tissues through which they pass, but X-rays that are refracted within those same tissues confound, rather than clarify, image contrast. The creation of contrast from the refraction of X-rays, rather than exclusively from absorption, yields images that display more detail with clearer distinction between tissue interfaces. Refraction-based imaging can reveal tiny structures that are transparent to X-ray attenuation but have sufficient variation in density to produce refraction contrast. Furthermore, refraction-based imaging decreases the required radiation dose.²¹To obviate the superimposing effects in a 2-dimensional DEI refraction image, we considered that combining CT with DEI would yield images with even greater clarity. CT allows a 3D representation of the sample, such that contrast from features at different depths are no longer superimposed on one another but can be separated and viewed as independent structures. Although this advantage is valuable in traditional absorption imaging, the additional features that provide contrast in a refraction-based image enhance the value of CT. Combining DEI technology, which is capable of imaging soft-tissue detail, with CT, which allows segregation of the contrast images at different depths, overcomes limitations of conventional X-ray imaging, namely lack of distinction of soft tissues and 2-dimensionality. As we report here, DEI combined with CT and a synchrotron-generated X-ray source yields 3D images of growing joint tissues at a resolution on the order of micrometers, which is much higher than can be generated using conventional imaging techniques.A synchrotron radiation source was required for the development of DEI because a synchrotron currently is the only source capable of providing an intensely brilliant light (millions of times brighter than sunlight and conventional X-ray sources), is highly collimated (light rays in the beam remain parallel with negligible dispersion over distance), can be made to be monochromatic (having a single wavelength), and can be tuned precisely to an array of energy ranges. The Canadian Light Source (www.lightsource.ca), which began operations in 2005, is one of only 47 synchrotron facilities worldwide and the only such facility in Canada. Although nonsynchrotron sources of X-rays for DEI–CT are conceivable,^16,18 such technology requires considerable image-acquisition time. Regardless, the quality of images generated by using synchrotron technology likely would remain the standard with which any new nonsynchrotron DEI–CT technological innovations would be compared.¹⁴Despite refinements in medical imaging, conventional radiography, CT scanning, and MRI still are insufficient to discern fine details, particularly in growing joints in which soft tissues (including cartilage) predominate and change with physiologic growth. The impetus for the current research was to develop an imaging technique that better demonstrated normal joint characteristics during growth and, in the future, could be applied to pathologic joints for experimental research and eventually clinical applications. In particular, we were motivated by a need to more effectively and reliably image growing joints affected by arthritis, a disease associated with alterations of bone and cartilage growth, tissue morphology and vascularity. Childhood arthritis research likely will benefit from having an improved imaging technique to aid in early diagnosis, monitor disease progression, and assess responses to therapies. The long-term outcomes of childhood arthritis are improved with early diagnosis and prompt and effective response to treatment interventions. Clinical and laboratory-based indicators of inflammation are not always adequate to detect and monitor subclinical intraarticular inflammation which, as with overt disease, can lead to progressive joint damage. Imaging can augment clinical and laboratory assessment of arthritis activity, but even the most sensitive currently available modalities are unable to detect all joint pathology.In juvenile arthritis, joint-imaging outcomes are difficult to evaluate because variations associated with normal growth cannot always be easily discerned from variations induced by the disease. Conventional radiography tends to detect advanced joint damage that has affected bone, but cartilage can be assessed only indirectly, and soft tissue abnormalities cannot be fully evaluated. Consequently, conventional radiography has insufficient sensitivity and specificity to be considered useful for diagnosing or monitoring children with inflammatory joint disease.^6,20 MRI, which evaluates both soft tissues and osteochondral structures, can be used to detect cartilage loss, bone erosions, and synovial hypertrophy in children and adolescents, and contrast-enhanced MRI detects active synovitis.^1,10 However, standardized approaches to acquire and interpret MRI data are not established for children in general and, in particular, for children with arthritis;^12,15 it is not always clear, for example, if observed thinning of cartilage is physiologic or pathologic. Furthermore, although MRI is more sensitive than conventional radiography, MRI too has limited precision in detecting fine structures and pathologic changes; a clinical MRI has less than 50% sensitivity in detecting cartilage damage that subsequently is seen arthroscopically.^8,13CT offers another option for joint visualization, given that it provides high-resolution, 3D images of bone from any angle. Despite its high spatial resolution, however, CT cannot match MRI''s soft-tissue contrast resolution, because CT provides negligible variability of attenuation coefficients of soft tissues so attenuation is nearly the same for cartilage, muscles, and ligaments. Furthermore, CT''s value is offset by the necessity for radiation exposure, a particular concern in the pediatric population. Therefore, for joint research and clinical applications, each of the conventional imaging techniques currently available has limitations. A safe, higher resolution imaging system that generates good contrast for all joint structures is required.Because the DEI technique initially was developed by using a synchrotron light source, we similarly used synchrotron technology in the current experiments. In contrast to conventional X-ray tubes, a synchrotron generates light by using radiofrequency waves and electromagnets to energize and accelerate electrons, thus producing brilliant, highly focused light from the entire wavelength spectrum, including X-rays. For the development and evaluation of DEI–CT imaging of joints, we chose to use healthy commercial piglet stifle joints because porcine stifle joints are anatomically similar to human knees.⁵ In addition, pigs grow quickly, reaching skeletal maturity at the distal femur and proximal tibia in 20 mo,¹⁹ thus allowing for the use of the pig as a model to study growth patterns in normal and disease states in a relatively short time period. The current study aimed to develop and test a new technology for imaging growing joints by using DEI combined with CT and a synchrotron radiation source. This report is the first to document the application of DEI–CT for imaging intact, growing joints.     

The cytoplasmic domain of tissue factor is phosphorylated by a protein kinase C-dependent mechanism.

Tissue factor (TF) is an integral membrane glycoprotein that serves as a cellular receptor and cofactor for the activation of the plasma protease factor VII. TF activity in both monocytes and endothelial cells is regulated by various cytokines and mitogens, including the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA). Three TF constructs (full-length human, a cytoplasmic domain deletion mutant, and a human-rat TF chimera), expressed in a human kidney cell line, were used to examine the in vivo phosphorylation state of TF after PMA treatment. The cytoplasmic domains of both rat and human TF were rapidly phosphorylated after cells were treated with 10-100 nM PMA. This response was completely abolished by preincubating cells with staurosporine, the potent PKC inhibitor, prior to PMA treatment. Localization of the phosphorylation site(s) to the cytoplasmic domain was demonstrated using a deletion mutant of TF and by CNBr digestion at the single methionine residue (Met-210) in the TF sequence. The rat TF cytoplasmic domain was phosphorylated to a higher specific activity than the human TF cytoplasmic domain. Phosphoamino acid analysis of the chimeric TF revealed both phosphothreonine and phosphoserine, whereas human TF contained only phosphoserine. Thus both potential phosphoacceptor sites are phosphorylated in the rat TF cytoplasmic domain. Alignment of TF cDNA sequences of mouse, rat, rabbit, and man revealed that the phosphoacceptor site (X-S*/T*-P-X, where asterisk indicates the phosphorylated residue) in the cytoplasmic domain has been conserved through evolution.     

Purification and characterization of a protein-tyrosine kinase from bovine thymus

A protein-tyrosine kinase has been isolated from a soluble extract of bovine thymus based on its ability to phosphorylate the tyrosine-containing peptide angiotensin I. The purification procedure employs sequential chromatography on columns of DEAE-cellulose, heparin-agarose, casein-agarose, butyl-agarose, and Sephadex G-75. The purified enzyme (p40) is a monomer of Mr = 40,000. The p40 kinase contains an ATP-binding site as determined by photoaffinity labeling experiments and catalyzes an intramolecular autophosphorylation reaction that leads to its modification on tyrosine. Of several proteins tested only the cytoplasmic domain of the erythrocyte band 3 protein serves as a good substrate for p40 (Km = 12 microM). Increasing concentrations of NaCl stimulate the phosphorylation of angiotensin I, inhibit the phosphorylation of band 3, and have no effect on the autophosphorylation of p40. At low concentrations of NaCl, Mn2+ is the preferred divalent cation. Peptide mapping experiments indicate that p40 is distinct from pp60src and from the major phosphotyrosine containing proteins of T and B lymphocyte membranes.     

Generation of an active protein-tyrosine kinase from lymphocytes by proteolysis

The major NaCl-stimulated protein-tyrosine kinase activity found in soluble thymus extracts, as measured by the phosphorylation of angiotensin I, is a 40-kDa enzyme known as p40 (Zioncheck, T. F., Harrison, M. L., and Geahlen, R. L. (1986) J. Biol. Chem. 261, 15637-15643). Antibodies prepared against p40 cross-react with a 72-kDa protein-tyrosine kinase (p72) from spleen or thymus that is closely related to p40 by peptide-mapping experiments. The recovery of p40 from spleen homogenates is reduced while the recovery of p72 is enhanced by the addition of high concentrations of leupeptin or soybean trypsin inhibitor to the homogenization media. The generation of p40 in spleen homogenates occurs with a concomitant increase in protein-tyrosine kinase activity. Activated catalytic fragments of 38-43 kDa can be generated by the treatment of partially purified p72 with trypsin or papain. The p72 protein-tyrosine kinase is found at the highest levels in spleen, thymus, and lung, tissues that also have high protein-tyrosine kinase activity and generate high levels of p40 following homogenization. p72 is also found in certain T and B cell-derived cell lines and in NIH3T3 cells.     

An anchored restriction-mapping approach applied to the genetic analysis of the Anopheles gambiae malaria vector complex 1

We introduce here a simple approach for rapidly determining restriction maps for a number of regions of a genome; this involves "anchoring" a map with a rare restriction site (in this case the seldom-cutting EagI) followed by partial digestion of a frequent-cutting enzyme (e.g., Sau 3A). We applied this technology to five species of the Anopheles gambiae complex. In a single Southern blot we obtained about a 15-kb restriction map each for the mtDNA, rRNA gene, and a scnDNA region for each of five species. Phylogenetic analyses of these regions yield trees at odds with the more traditional chromosome inversion-based trees. The value of the approach for systematic purposes is the ease with which several large, independent regions of the genome can be quickly assayed for molecular variation.      

Interaction of apolipoprotein(a) with apolipoprotein B-containing lipoproteins

Recombinant DNA-derived apolipoprotein(a) was used to demonstrate that the apo(a) moiety of lipoprotein(a) (Lp(a)) is responsible for the binding of Lp(a) to other apolipoprotein B-containing lipoproteins (apoB-Lp) including LDL2, a subclass of low density lipoproteins (d = 1.030-1.063 g/ml). The r-apo(a).LDL2 complexes exhibited the same binding constant as Lp(a).LDL2 (10(-8) M). Treatment of either recombinant apo(a) or Lp(a) with a reducing agent destroyed binding activity. A synthetic polypeptide corresponding to a portion of apo(a)'s kringle-4 inhibited the binding (K1 = 1.9 x 10(-4) M) of LDL2 to Lp(a). Therefore, we concluded that binding to apoB-Lp was mediated by the kringle-4-like domains on apo(a). Using ligand chromatography which can detect complexes having a KD as low as 10(-2) M, we demonstrated the binding of plasminogen to apoB-Lp. Like Lp(a), binding of plasminogen to apoB-Lp was mediated by the kringle domain(s). The differences in binding affinity may be due to amino acid substitutions in the kringle-4-like domain. In most of the kringle-4-like domains of apo(a), the aspartic residue critical for binding to lysine was substituted by valine. Consistent with this substitution, we found that L-proline and hydroxyproline, but not L-lysine, inhibited the binding of LDL2 to apo(a). Inhibition by L-proline could be reversed in the binding studies by increasing the amount of apo(a); and L-proline-Sepharose bound plasma Lp(a), suggesting that L-proline acted as a ligand for the kringle-4-like domain(s) of apo(a) involved in the binding of apoB-Lp. The binding of apo(a) to proline and hydroxyproline could be responsible for the binding of apo(a) to the subendothelial extracellular matrix, i.e. domains of proteins rich in proline or hydroxyproline (e.g. collagen and elastin).     

The relationship between codon boundaries and multiple reading-frame preferences: coding organization of bacterial insertion sequences

Theoretical considerations have shown that the five possible overlapping reading-frame configurations differ significantly in their coding flexibility and thus in their information content (Siegel and Fitch 1980; Smith and Waterman 1980). Contrary to expectation, the overlapping frame configuration allowing the greatest coding flexibility is rarely seen, whereas one of the most constraining is common. We point out here that this overlapping reading-frame paradox and an observed but unexplained preference in coding regions for a pyrimidine-purine at codon boundaries (Shepherd 1981; Jones and Kafatos 1982; Smith et al. 1983) are intimately linked. The codon boundary preference, which may be related to translation efficiency or accuracy, places constraints on the evolution of overlapping coding regions. These considerations may help identify actual coding regions in DNA sequences. We have analyzed five sequenced (enteric) bacterial insertion sequences for codon boundary incidences and reading-frame configurations and find that they are consistent with these proposed constraints.      

Vascular endothelial growth factor governs endothelial nitric-oxide synthase expression via a KDR/Flk-1 receptor and a protein kinase C signaling pathway.

The mechanism by which vascular endothelial growth factor (VEGF) regulates endothelial nitric-oxide synthase (eNOS) expression is presently unclear. Here we report that VEGF treatment of bovine adrenal cortex endothelial cells resulted in a 5-fold increase in both eNOS protein and activity. Endothelial NOS expression was maximal following 2 days of constant VEGF exposure (500 pM) and declined to base-line levels by day 5. The elevated eNOS protein level was sustained over the time course if VEGF was co-incubated with L-N(G)-nitroarginine methyl ester, a competitive eNOS inhibitor. Addition of S-nitroso-N-acetylpenicillamine, a nitric oxide donor, prevented VEGF-induced eNOS up-regulation. These data suggest that nitric oxide participates in a negative feedback mechanism regulating eNOS expression. Various approaches were used to investigate the role of the two high affinity VEGF receptors in eNOS up-regulation. A KDR receptor-selective mutant increased eNOS expression, whereas an Flt-1 receptor-selective mutant did not. Furthermore, VEGF treatment increased eNOS expression in a KDR but not in an Flt-1 receptor-transfected porcine aorta endothelial cell line. SU1498, a selective inhibitor of the KDR receptor tyrosine kinase, blocked eNOS up-regulation, thus providing further evidence that the KDR receptor signals for eNOS up-regulation. Finally, treatment of adrenal cortex endothelial cells with VEGF or phorbol ester resulted in protein kinase C activation and elevated eNOS expression, whereas inhibition of protein kinase C with isoform-specific inhibitors abolished VEGF-induced eNOS up-regulation. Taken together, these data demonstrate that VEGF increases eNOS expression via activation of the KDR receptor tyrosine kinase and a downstream protein kinase C signaling pathway.     

Evolutionary relatedness of some primate models of Plasmodium

Primate--and, specifically, monkey--malaria infections are commonly used for understanding the pathology of and immune response to the human disease because they are thought to resemble most closely the host-parasite relationship found in humans. Plasmodium cynomolgi is used extensively as a model for the human parasite, P. vivax, and P. knowlesi is used primarily as a model for the development of erythrocytic-stage vaccines. Both of these simian parasites can naturally infect man, resulting in mildly symptomatic episodes of the disease. The phylogenetic relationship between these two simian parasites and previously characterized Plasmodium species, including P. vivax, was examined by comparison of the asexually expressed small- subunit ribosomal RNA genes. Our analysis confirmed that P. vivax is most closely related to P. cynomolgi and that it remains an appropriate model of the human pathogen. Furthermore, with P. knowlesi and P. fragile, these two species form a group of closely related species, distant from other Plasmodium species. What is considered to be the most ancient of the human malaria pathogens, P. malariae, was also included in the analysis and does not group at all with other simian or human parasites.