Reciprocal relation between VEGF and NO in the regulation of endothelial integrity.

Balloon angioplasty disrupts the protective endothelial lining of the arterial wall, rendering arteries susceptible to thrombosis and intimal thickening. We show here that vascular endothelial growth factor (VEGF), an endothelial cell mitogen, is upregulated in medial smooth muscle cells of the arterial wall in response to balloon injury. Both protein kinase C (PKC) and tyrosine kinase pp60src mediate augmented VEGF expression. In contrast, nitric oxide (NO) donors inhibit PKC-induced VEGF upregulation by interfering with binding of the transcription factor activator protein-1 (AP-1) to the VEGF promoter. Inhibition of VEGF promoter activation suggests that NO secreted by a restored endothelium functions as the negative feedback mechanism that downregulates VEGF expression to basal levels. Administration of a neutralizing VEGF antibody impaired reendothelialization following balloon injury performed in vivo. These findings establish a reciprocal relation between VEGF and NO in the endogenous regulation of endothelial integrity following arterial injury.

Clinical potential of intravascular ultrasound for physiological assessment of coronary stenosis: relationship between quantitative ultrasound tomography and pressure-derived fractional flow reserve.

BACKGROUND: Little is known regarding intravascular ultrasound (IVUS) criteria to determine the functional severity of coronary stenosis. Recently, fractional flow reserve (FFR) has emerged as a lesion-specific index of the functional severity of a coronary stenosis that is independent of systemic hemodynamic variability. The present study was undertaken to determine the IVUS parameters for the physiological severity of coronary stenosis. METHODS AND RESULTS: Fifty-one lesions in 42 patients were evaluated by means of quantitative coronary angiogram, IVUS, and intracoronary pressure measurements. The FFR was calculated as the ratio of the distal coronary pressure divided by the proximal coronary pressure under hyperemia. We considered a value of the FFR <0.75 as significant in determining inducible ischemia, according to the previous studies. The minimal luminal area (MLA) and the area stenosis were measured by IVUS. By regression analysis, the MLA showed a positive correlation with the FFR value (r(2)=0.62, P<0.0001). The area stenosis had a significant inverse correlation with the value of FFR (r(2)=0.60, P<0.0001). The IVUS thresholds that maximized the sensitivity and specificity were MLA <3.0 mm(2) (sensitivity, 83.0%; specificity, 92.3%) and area stenosis >0.6 (sensitivity, 92.0%; specificity, 88.5%). The combination of both criteria (MLA <3.0 mm(2) and area stenosis <0.6) without exception met a value of the FFR <0.75. CONCLUSIONS: Anatomic parameters obtained by IVUS showed a significant correlation to the FFR values. The present study demonstrated that the combination of the MLA and area stenosis measured by IVUS can be an anatomic predictor for the physiological impact of coronary epicardial stenosis.

Influence of plasma lipoprotein (a) levels on coronary vasomotor response to acetylcholine.

OBJECTIVES: This study was undertaken to examine the influence of plasma lipoprotein(a) [Lp(a)] levels on coronary endothelial vasomotor function. BACKGROUND: Epidemiologic studies have demonstrated a direct relation between elevated plasma levels of Lp(a) and increased risk of coronary artery disease. Well recognized coronary risk factors are known to affect endothelium-dependent vasomotion; however, the influence of Lp(a) on coronary vasomotor function has not been determined. METHODS: We used quantitative coronary angiography to measure left anterior descending coronary artery diameter changes produced by intracoronary acetylcholine and isosorbide dinitrate in 30 patients with angiographically normal coronary arteries. Plasma Lp(a) levels were determined with enzyme-linked immunosorbent assay. RESULTS: Vasomotor response to acetylcholine ranged from +13% to -47% in the proximal, from +23% to -53% in the middle and from +13% to -56% in the distal segment of the left anterior descending coronary artery. According to univariate linear regression analysis, Lp(a) had a significant inverse correlation with vasomotor response to acetylcholine: r = 0.47, p < 0.01 in the proximal; r = -0.61, p < 0.001 in the middle; and r = -0.52, p < 0.01 in the distal segment of the left anterior descending coronary artery. By multiple stepwise regression analysis, plasma Lp(a) was the significant predictor of vasomotion in response to acetylcholine in all tested segments (p < 0.01). CONCLUSIONS: Elevated Lp(a) levels were associated with impairment of endothelium-dependent vasodilation even when atherosclerotic lesions were not recognizable by angiography. This finding suggests that elevated plasma levels of Lp(a) cause endothelial dysfunction and may contribute in part to later development of atherosclerosis, as shown in epidemiologic studies.

Histochemical staining following LacZ gene transfer underestimates transfection efficiency.

Analysis of LacZ gene expression is conventionally inferred from blue staining that results from exposure of the transfected cells or tissue to the substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). Such histochemical staining reports not whether the gene product is present or absent, but where it is active. We investigated the hypothesis that identification of activity, as opposed to presence, of the enzyme underestimates gene expression following LacZ gene transfer. Under conditions optimized for in vitro histochemistry, up to 20% of cells stably transfected with nls-LacZ remained unstained by X-Gal. In contrast, immunostaining with a monoclonal or a polyclonal anti-beta-galactosidase (beta-Gal) antibody positively stained 99% of the cell nuclei. Following in vivo transfection of naked DNA encoding for nls-LacZ, X-Gal staining disclosed 2.7 +/- 1.7 positive nuclei per LacZ-transfected animal, or a transfection efficiency of 0.015%. In contrast, immunohistochemical staining disclosed 118 +/- 32.7 positive nuclei per transfected animal, yielding a transfection efficiency of 0.64% (p < 0.0001 versus X-Gal staining). Thus, 42.9 times more positive cells were detected by antibody than X-Gal staining. Finally, LacZ gene expression following intramuscular gene transfer with an adenoviral vector was observed in 7.6% of skeletal muscle cells assessed with X-Gal; anti-beta-Gal antibody identified 21.8% of cells as being successfully transfected (p < 0.0001). Thus, X-Gal histochemistry following gene transfer of constructs encoding LacZ may underestimate the anatomic extent of gene expression. The superior sensitivity of immunostaining suggests that anti-beta-Gal antibody represents the preferred analytical tool for light microscopic evaluation of LacZ gene transfer.

18F-fluoro-2'-deoxyuridine as a tracer of nucleic acid metabolism in brain tumors.

The value of 18F-fluoro-2'-deoxyuridine (18F-FUdR) as a tracer for nucleic acid metabolism was studied using an experimental rat brain-tumor model. The 18F activity in the tumor tissue 45 minutes after intravenous injection of 18F-FUdR was about 12 times higher than that in the contralateral cortex. Double-labeled autoradiography with 18F-FUdR and 14C-thymidine revealed similar brain-tumor images. In contrast, an autoradiographic comparison of 18F-FUdR with 14C-aminoisobutyric acid, which reveals the impairment of the blood-brain barrier, showed very different images. Also, the 18F radioactivity in the tumor tissue was at a constant level for 30 to 120 minutes, whereas a notable increase in 18F activity with time was observed in nucleotides and acid-insoluble fractions. These results suggest that the distribution pattern of 18F-FUdR closely correlates with the metabolism of nucleic acid and that this drug could be a useful tracer for positron emission tomography.

Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients (9). Evaluation of cytotoxicity test on HeLa cells.

The cytotoxicity test using MTT on HeLa cells (HeLa-MTT) was evaluated as an alternative method to the Draize eye irritation test (Draize test) by six to eight laboratories. The 50% inhibition concentration (EC(50)) for MTT reduction was calculated. A total of thirty-nine test chemicals were examined. The average interlaboratory coefficient of variation (CV) of the present assay was 25%. Comparison of the in vitro test results with those of the Draize test revealed a correlation coefficient of -0.799 between the log(EC(50)) value and the maximum average total score (MAS) for a 10% solution or suspension. The following characteristics of HeLa-MTT have become apparent through this validation: (1) HeLa-MTT could be applied to all substances including water-insoluble substances, esters, a colour additive, and a substance which is known to directly reduce MTT; (2) there is good interlaboratory reproducibility and strong correlation between HeLa-MTT and Draize MAS; (3) results for strong acids, alkanolamines and alcohols (lower mono-ol) clearly deviate from other samples with respect to the correlation between HeLa-MTT and Draize MAS. These results suggest that HeLa-MTT may be useful for predicting the Draize MAS if definite criteria can be established for applicable compounds.

WS75624 A and B, new endothelin converting enzyme inhibitors isolated from Saccharothrix sp. No. 75624. II. Structure elucidation of WS75624 A and B.

The structures of WS75624 A and B, novel endothelin converting enzyme inhibitors, were determined to be 1 and 2A, respectively, by a combination of chemical evidence and a series of 2D NMR spectral analyses.

Chaetiacandin, a novel papulacandin. I. Fermentation, isolation and characterization.

Chaetiacandin, a new anti-yeast antibiotic structurally related to papulacandin, was isolated from a culture of Monochaetia dimorphospora. The fermentation, isolation, and physico-chemical and biological properties are reported. The molecular formula of this compound was determined as C43H60O16.

Biological and pharmacological properties of highly selective new endothelin converting enzyme inhibitor WS79089B isolated from Streptosporangium roseum No. 79089.

WS79089B a highly specific endothelin converting enzyme (ECE) inhibitor has been isolated from the fermentation broth of Streptosporangium roseum No. 79089. WS79089B showed highly selective ECE inhibition activity with IC50 value of 0.14 microM and behaved as a competitive inhibitor of ECE, with Ki values of 8.9 x 10(-8) M. The sodium salt of WS79089B (FR901533) inhibited big endothelin-1 (big ET-1) induced pressor effect in a dose dependent manner when administered to male Sprague-Dawley rats intravenously dosed 2 minutes prior to big ET-1 challenge.

Studies on WF-3681, a novel aldose reductase inhibitor. I. Taxonomy, fermentation, isolation and characterization.

WF-3681 was isolated from a cultured filtrate of Chaetomella raphigera as a novel inhibitor of aldose reductase. It was extracted with ethyl acetate and then purified with silica gel chromatography. Its molecular formula was determined to be C13H12O5 by elemental analysis and high resolution electron impact mass spectrometry. IC50 of WF-3681 was 2.5 X 10(-7) M for partially purified aldose reductase of rabbit lens.

FR191512, a novel anti-influenza agent isolated from a fungus strain No.17415. I. Taxonomy, fermentation, isolation, physico-chemical properties and structure elucidation.

In the course of our screening for anti-influenza agents of microbial origin, FR191512 was isolated from the cultured broth of fungus strain No. 17415 as colorless powder. The structure of FR191512 was determined by several spectroscopic experiments as a novel polyphenolic compound. This compound showed potent antiviral activity against influenza A virus.

WS79089A, B and C, new endothelin converting enzyme inhibitors isolated from Streptosporangium roseum. No. 79089. Taxonomy, fermentation, isolation, physico-chemical properties and biological activities.

WS79089A, B and C, which are novel endothelin converting enzyme (ECE) inhibitors have been isolated from the fermentation broth of Streptosporangium roseum No. 79089. These inhibitors were purified from an acetone extract of whole culture broth followed by Silicar CC-4 column chromatography and HPLC. WS79089A, B and C showed highly selective ECE inhibition activity with IC50 values of 0.73 microM 0.14 microM and 3.42 microM, respectively. On the basis of spectroscopic and chemical evidence, the tentative structures of WS79089A, B and C have been proposed, they have benzo[a]naphtacen chromophores.

WF11605, an antagonist of leukotriene B4 produced by a fungus. I. Producing strain, fermentation, isolation and biological activity.

WF11605, a new antagonist of leukotriene B4 (LTB4) was isolated as a product of fungal strain F11605. The molecular formula of WF11605 was determined to be C38H60O11. WF11605 inhibited LTB4-induced chemotaxis of rabbit polymorphonuclear leukocytes (PMNLs) with an IC50 value of 1.7 x 10(-7) M and blocked 3H-LTB4 binding to PMNL membranes at 5.6 x 10(-6) M (IC50). WF11605 also inhibited LTB4-induced degranulation of rabbit PMNLs at 3.0 x 10(-6) M (IC50). However, WF11605 did not show any inhibitory effect on platelet activating factor (PAF)- and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-induced degranulation at concentrations up to 10(-4) M. These results suggest that WF11605 is a specific antagonist of LTB4.

Vinigrol, a novel antihypertensive and platelet aggregation inhibitory agent produced by a fungus, Virgaria nigra. I. Taxonomy, fermentation, isolation, physicochemical and biological properties.

Vinigrol, produced by a fungal strain identified as Virgaria nigra, was extracted from the cultured mycelium, purified by solvent extraction followed by chromatography on silica gel and then isolated as crystals (C20H34O3, mp 108 degrees C). Vinigrol decreased arterial blood pressure of anesthetized normotensive rats dose-dependently when administered intravenously. Vinigrol inhibited platelet activating factor and epinephrine induced platelet aggregation.

A new immunomodulator, FR-900490.

FR-900490 is a new type of immunoactive substance produced by a fungus Discosia sp. F-11809. The colony forming units in culture (cfu-c) in bone marrow cells, which were suppressed by immunosuppressive factor obtained from the serum of sarcoma 180 tumor bearing mouse, was restored to normal level by the addition of FR-900490 in vitro. Furthermore, in mitomycin C (MMC)-treated mice the subsequent administration of FR-900490 caused a significant increase of cfu-c in bone marrow cells depressed by MMC.

A new immunomodulator, FR-900483.

FR-900483 is a new immunoactive substance produced by a fungus, Nectria lucida F-4490. Concanavalin A-stimulated lymphocyte proliferation, which had been suppressed by addition of immunosuppressive factor, was restored to a normal level by the addition of FR-900483. Furthermore, FR-900483 restored the capacity of immunosuppressed mice to produce antibody against sheep red blood cells.

A new angiogenesis inhibitor, FR-111142.

FR-111142 is a new angiogenesis inhibitor produced by a fungus Scolecobasidium arenarium F-2015. FR-111142 inhibited endothelial cell proliferation in vitro and angiogenesis in the growing chick chorioallantoic membrane model in vivo. Further, FR-111142 also suppressed the solid tumor growth in mice.

Physiological analysis of bicozamycin high-producing Streptomyces griseoflavus used at industrial level.

Streptomyces griseoflavus, a bicozamycin-producing wild type strain and its high-producing one derived from it by multiple (greater than 15) mutagenic treatments, were analyzed physiologically and biochemically. The high-producing strain was characterized by: (1) An increased pool size of amino acids including leucine and isoleucine, precursors for bicozamycin synthesis, (2) an earlier and greater accumulation of intracellular ppGpp, (3) a more accentuated decrease in GTP pool size, (4) a higher specific activity of ornithine transcarbamylase which produces citrulline, (5) an increased ability to form aerial mycelium, and (6) an increased resistance to its own antibiotic. We propose that (1), (2) and (4) may be responsible for the high yields of bicozamycin and, possibly, of some other antibiotics produced by Streptomyces sp.

FR65814, a novel immunosuppressant isolated from a Penicillium strain. Taxonomy, fermentation, isolation, physico-chemical and biological characteristics and structure assignment.

FR65814, a novel immunosuppressant, was isolated from the cultured broth of Penicillium jensenii F-2883. The structure was assigned to be 5,6-dihydroxy-4-(1,2-epoxy-1,5-dimethyl-4-hexenyl)-1-oxaspiro++ +[2,5]octane by spectroscopic analyses. The compound suppressed the immune response at low concentration. In addition, a structually related component fumagillol, a known carcinolytic agent, was also isolated and found to show immunosuppressive activity.

Chryscandin, a novel peptidyl nucleoside antibiotic. I. Taxonomy, fermentation, isolation and characterization.

A novel antifungal antibiotic, chryscandin was found in the culture broth of a strain of fungi. The producing organism was subsequently identified as Chrysosporium pannorum. The antibiotic obtained as colorless needle crystals (C20H23N7O6, MW 457) is active against Candida albicans and Gram-positive bacteria, but it is ineffective against filamentous fungi and Gram-negative bacteria. Acute toxicity is very low in mice.