Advances in imaging angiogenesis and inflammation in atherosclerosis.

Advances in imaging technology have provided powerful tools for dissecting the angiogenic and inflammatory aspects of atherosclerosis. Improved technology along with multi-modal approaches has expanded the utilisation of imaging. Recent advances provide the ability to better define structure and development of angiogenic vessels, identify relationships between inflammatory mediators and the vessel wall, validate biological effects of anti-inflammatory and anti-angiogenic drugs, delivery and/or targeting specific molecules to inflammatory regions of atherosclerotic plaques.

Inhibitor of DNA binding-4 promotes angiogenesis and growth of glioblastoma multiforme by elevating matrix GLA levels.

Inhibitor of differentiation-4 is highly expressed in glioblastoma multiforme (GBM). We report a novel pro-angiogenic function for inhibitor of differentiation-4 in the growth of glioblastoma xenografts. Tumor-derived cell cultures expressing elevated levels of ID4 produced enlarged xenografts in immunosuppressed mice that were better vascularized than corresponding control tumors and expressed elevated matrix GLA protein (MGP) that mediated enhanced tumor angiogenesis. Inhibition of MGP resulted in smaller and less vascularized xenografts. Our finding shows a novel function for ID4 in tumor angiogenesis, and identifies ID4 and MGP as possible therapeutic targets for GBM.

Plasminogen and plasmin activity in patients with coronary artery disease.

OBJECTIVE: While coronary artery disease (CAD) is associated with disturbances of the plasma fibrinolytic system, the nature of these disturbances is not fully defined. Fibrinolysis is regulated by plasmin, whose production is mediated by plasminogen activator conversion of plasminogen (Plg) to plasmin. The cascade is modulated by feedback loops that include Plg activator inhibitor 1 (PAI-1). Molecular interactions with Plg kringle domains play an important role in regulating plasmin production and its modulation of fibrinolysis. We hypothesized that interactions of tissue plasminogen activator (tPA) with Plg kringle domains regulates plasmin levels in patients with stable CAD. METHODS: Plasma was collected from patients (n = 33) with an angiographically significant CAD and controls (n = 18) with angiographically established normal or minimally diseased arteries. Plasmin activity, tPA activity, and plasma levels of Plg, PAI-1, uPA, and tPA were determined. RESULTS: CAD patients had 1.7-fold greater plasmin activity (P = 0.02) that correlated with 1.5-fold higher tPA activity when compared to controls. Epitope mapping of Plg domains showed Plg differences in epitope exposure between the two groups. Plasma from CAD patients had 50% less (P < 0.001) detectable kringle 4 and 48% less (P = 0.007) detectable kringles 1-3. CONCLUSIONS: Based on detectable differences in Plg, we conclude that in patients with stable CAD, Plg complexed with tPA exists in a conformation that enables increased tPA activity and Plg conversion to plasmin.

A truncated plasminogen activator inhibitor-1 protein induces and inhibits angiostatin (kringles 1-3), a plasminogen cleavage product.

Plasminogen activator inhibitor-1 (PAI-1) is a serpin protease inhibitor that binds plasminogen activators (uPA and tPA) at a reactive center loop located at the carboxyl-terminal amino acid residues 320-351. The loop is stretched across the top of the active PAI-1 protein maintaining the molecule in a rigid conformation. In the latent PAI-1 conformation, the reactive center loop is inserted into one of the beta sheets, thus making the reactive center loop unavailable for interaction with the plasminogen activators. We truncated porcine PAI-1 at the amino and carboxyl termini to eliminate the reactive center loop, part of a heparin binding site, and a vitronectin binding site. The region we maintained corresponds to amino acids 80-265 of mature human PAI-1 containing binding sites for vitronectin, heparin (partial), uPA, tPA, fibrin, thrombin, and the helix F region. The interaction of "inactive" PAI-1, rPAI-1(23), with plasminogen and uPA induces the formation of a proteolytic protein with angiostatin properties. Increasing amounts of rPAI-1(23) inhibit the proteolytic angiostatin fragment. Endothelial cells exposed to exogenous rPAI-1(23) exhibit reduced proliferation, reduced tube formation, and 47% apoptotic cells within 48 h. Transfected endothelial cells secreting rPAI-1(23) have a 30% reduction in proliferation, vastly reduced tube formation, and a 50% reduction in cell migration in the presence of VEGF. These two studies show that rPAI-1(23) interactions with uPA and plasminogen can inhibit plasmin by two mechanisms. In one mechanism, rPAI-1(23) cleaves plasmin to form a proteolytic angiostatin-like protein. In a second mechanism, rPAI-1(23) can bind uPA and/or plasminogen to reduce the number of uPA and plasminogen interactions, hence reducing the amount of plasmin that is produced.

Inhibition of cytoplasmic antigen, glucose- 6-phosphate dehydrogenase, by VH-CH1, an intracellular Fd fragment antibody derived from a semisynthetic Fd fragment phage display library.

A library of Fd fragment antibody binding proteins was created by random mutation of 15 nucleotides within the CDRIII region of the immunoglobulin heavy chain gene and displayed as Fd coat protein fusion constructs of M13 phage. The library was screened for those VHbinding sites that bound glucose-6-phosphate dehydrogenase (G6PD). One isolate (DH27bp) inhibited G6PD activity by 85 %. The DH27bpgene was re-engineered, placed in a eukaryotic expression vector having an isopropyl-beta-delta-thiogalactopyranoside (IPTG) inducible promoter, and transfected and then expressed in Chinese hamster V79 cells. G6PD activity was completely inhibited. Removal of IPTG reverted the cell to full G6PD activity. The intracellular dynamics of the G6PD/DH27bpcomplex showed that when the proteasomes of cells expressing DH27bpwere inhibited (N -acetyl-Leu-Leu-norleucinal or lactacystin) G6PD activity increased. Metabolic labelling of newly synthesized IPTG-induced proteins during/absence of proteasomal inhibitors showed that both G6PD and DH27bpare signaled for degradation when the intracellular complex is formed. Furthermore, semi-quantitative RT/PCR demonstrated that G6PD mRNA is upregulated over the time course of G6PD inactivation by DH27bpFd binding protein. These effects were not observed in those cells expressing a non-mutated Fd (UMHC) or in IPTG-treated non-transduced V79 cells. Our results demonstrate that an Fd-based intracellular binding protein can find and disable the function of a specific intracellular target and once the Fd expression is repressed the activity of intracellular targeted protein can revert to normal.

The effect of acetaminophen, ibuprofen, and misoprostol on prostaglandin E2 synthesis and the degree and rate of orthodontic tooth movement.

The present study compared the effect of acetaminophen, ibuprofen and misoprostol on PGE2 synthesis and orthodontic tooth movement. Guinea pigs were randomly assigned into one of three test groups or a control group. Each group received study treatments every 12 hours as an orthodontic force was applied to the maxillary incisors. Direct linear measurements of tooth separation were recorded at days 2, 4, 6, 10, and 11, and inflammatory exudate from the periodontal ligament (PDL) space was extracted and quantitatively analyzed radioimmunologically for the presence of PGE2 at days 4 and 9. Comparing the concentration of PGE2 in sample extracts, a significant difference (P = 0.001) was found among drug groups. A highly significant difference was found between the mean tooth separation among the various drug groups (P < 0.001). At day 11 the misoprostol group exhibited 4.49 +/- 0.49 mm of separation; ibuprofen 2.56 +/- 0.11 mm, and the control and acetaminophen groups exhibited similar degrees of tooth separation: 3.31 +/- 0.07 mm and 3.31 +/- 0.08 mm, respectively. A highly significant difference occurred between the mean rates of tooth separation among the various drug groups after day 8 (P < 0.001). Results of this study suggest that acetaminophen is the analgesic of choice for the relief of minor discomfort associated with orthodontic treatment.

The effect of different debonding techniques on the enamel surface: an in vitro qualitative study.

The purpose of this in vitro study was to evaluate the enamel surface structure subjected to various techniques of debonding orthodontic attachments and to develop a technique for residual adhesive removal that restores the enamel surface as closely as possible to its pretreatment condition without introducing iatrogenic damage. Enamel surface structure was examined with a scanning electron microscope before bonding of twin metal brackets to 60 previously extracted premolars with two heavily filled composite resins. Two groups, each consisting of 30 teeth, were equally subdivided into 10 subgroups. The first three subgroups were used to compare the efficacy of three bracket removing instruments. Since there were no differences in the debonding properties between the two resins, the two groups were combined. In this way, each subgroup (n = 6) could be used for a more meaningful comparison. On the basis of the results of this comparison, the bracket removing instrument that produced the most consistent separation at the bracket-adhesive interface was used in the remainder of the study. After appliance removal, the teeth were again examined microscopically and photographed, and seven different procedures for residual resin removal were compared. After resin removal, the final polished enamel surface was followed by microscopic evaluation. Results of this study show the bracket removing plier produced the most consistent separation at the bracket-adhesive interface, leaving the enamel surface intact. Carbide burs at high speed and air coolant proved to be efficient in residual resin removal, but when used alone, failed to produce a satisfactory enamel surface. After the removal of residual resin, graded medium, fine, and superfine Sof-Lex finishing disks (Unitek Corp., Monrovia, Calif.) produced surfaces that could be readily restored satisfactorily after receiving a final polish with a rubber cup and Zircate paste.

Synthesis, antitumor activity, and antiviral activity of 3-substituted 3-deazacytidines and 3-substituted 3-deazauridines.

Novel 3-substituted analogues of 4-amino-1-beta-D-ribofuranosyl-2(1H)-pyridinone (3-deazacytidine, 3) and 4-hydroxy-1-beta-D-ribofuranosyl-2(1H)-pyridinone (3-deazauridine, 4) have been synthesized and tested for antitumor and antiviral activity. Thus the 3-chloro (9a), 3-bromo (9b), and 3-nitro (9c) analogues of 3 and the 3-chloro (9d), 3-bromo (9e), and 3-nitro (9f) analogues of 4 were prepared by standard glycosylating procedures. Novel requisite heterocycles 4-amino-3-chloro-2(1H)-pyridinone (7a) and 4-amino-3-bromo-2(1H)-pyridinone (7b) were prepared by halogenating 4-amino-2(1H)-pyridinone (5). Requisite heterocycles 4-amino-3-nitro-2(1H)-pyridinone (7c), 3-chloro-4-hydroxy-2(1H)-pyridinone (7d), 3-bromo-4-hydroxy-2(1H)-pyridinone (7e), and 4-hydroxy-3-nitro-2(1H)-pyridinone (7f) were synthesized by known procedures from 4-hydroxy-2(1H)-pyridinone (6). Structure proof of target nucleosides was provided by independent synthesis, 1H NMR, and UV. Compounds 9a-f were devoid of activity against intraperitoneally implanted L1210 leukemia in mice. Compound 9f displayed significant activity against rhinovirus type 34 grown in WISH cells. 4-Amino-3-fluoro-1-beta-D-ribofuranosyl-2(1H)-pyridinone (1) displayed good activity against intraperitoneally implanted P388 leukemia in mice, but it was devoid of activity against M5076 sarcoma, amelanotic (LOX) melanoma xenograft, and subrenal capsule human mammary carcinoma MX-1 xenograft in mice. Compound 1 also displayed significant activity against rhinovirus type 34.

Antiviral and cytotoxicity evaluation of 3-nitro-3-deazauridine.

3-Nitro-3-deazauridine (3N-3DU) is a new synthetic nucleoside having activity against members of 5 RNA virus families including: paramyxoviruses (parainfluenza, PIV), picornaviruses (rhino-, RV), rhabdoviruses (vesicular stomatitis, VSV), togaviruses (Semliki Forest, SFV) and bunyaviruses (Punta Toro, PTV). In this report, we evaluate and compare its activity with the parent nucleoside, 3-deazauridine (3DU) and ribavirin as drug standards. Comparison of drug activities utilizes observations of antiviral indices, which are determined by the following formula: maximum tolerated dose (MTD)/minimum inhibitory concentration (MIC). The antiviral index (AI) of 3N-3DU (AI 15.3) was comparable to ribavirin and much higher than 3DU when evaluated against PIV. The 3N-3DU was the most active of the three when tested against RV (AI 24.1), SFV (AI 76.9) or VSV (AI 50). In contrast to the RV activity, 3N-3DU (AI 0.5) and 3DU (AI less than 0.1) were less active than ribavirin (AI 1.3) when evaluated against poliovirus, type 1 (PoV). Ribavirin (AI 10.0) was more active than 3N-3DU (AI 2.4) and 3DU (AI less than 0.1) against PTV. 3N-3DU exhibited comparable toxicity to ribavirin in KB cells, was 4-fold less toxic in WISH cells and 4-fold more toxic in LLC-MK2 cells. Overall, 3N-3DU is markedly less toxic than its parent nucleoside, 3DU. It appears from this study that the structural modification of 3DU resulting from the addition of the nitro group in the 3 position of the base reduces toxicity and enhances the antiviral activity.

A simple method of drying virus on inanimate objects for virucidal testing.

A simple method for drying virus on inanimate objects (cover slips) under vacuum in the cold is described. Following this procedure virus maintains high titers (10(6-7)) for periods of 1-3 wk at -70 degrees C depending on the virus. For virucidal assay of disinfectants, cover slips are exposed to medium simulating the disinfectant (virus control) or disinfectant in an upright position in an Ultra-Vu cuvette. Cover slips are readily removed and placed in tissue culture medium for dilution of virus and determination of virus titer. Cytotoxicity of disinfectant is determined by exposing cover slip without virus to disinfectant, then placing it in medium, diluting the medium and incubating with the indicator cells. The use of this technique results in high titers of virus on cover slips, which are inanimate objects requiring minimal manipulation. The titration of virus or cytotoxicity in microplates is cell, medium, serum, and labware economical.