Masako Yasuda

Stimulation of in vitro angiogenesis by hydrogen peroxide and the relation with ETS-1 in endothelial cells.

The purpose of this study was to examine the effect of hydrogen peroxide (H2O2) on angiogenesis in cultured endothelial cells. Endothelial cells obtained from bovine thoracic aorta (BAECs) were cultured between two layers of collagen type I to measure the tube formation which is a marker for angiogenesis. Addition of H2O2 (0.1-10 microM) to endothelial cells for various periods increased the rate of tube formation. The maximum stimulation of the tube formation was obtained when cells were exposed to 1 microM H2O2 for 30 min, and the enhancement of tube formation was blocked by catalase (10 U/ml). Both proliferation and migration of BAEC which are known to affect angiogenesis, were also stimulated by the addition of H2O2 (0.1 and 1 microM). Thus relatively low concentrations of H2O2 stimulated angiogenesis, proliferation and migration. Ets-1 is a member of the ets gene family of transcription factors, which binds to the ets binding motif in the cis-acting elements and regulates the expression of certain genes such as proteases including urokinase plasminogen activator (u-PA) and matrix metalloproteinase-1 (MMP-1). Interestingly, H2O2 increased the ets-1 mRNA level in BAECs compared with the basal level. The H2O2-stimulated angiogenesis was completely blocked by an ets-1 antisense oligonucleotide, but not by a mismatched oligonucleotide. These findings indicate that low concentrations of H2O2 stimulate angiogenesis in BAECs, and the stimulation mechanisms may partially involve the enhancement of proliferation and migration. Moreover, the H2O2-induced angiogenesis is likely to be mediated by the transcription factor ets-1.

A novel effect of polymorphonuclear leukocytes in the facilitation of angiogenesis

The purpose of this study was to examine whether the adhesion of polymorphonuclear leukocytes (PMNs) to endothelial cells and/or reactive oxygen species (ROS) released from PMNs are responsible for inducing angiogenesis. Angiogenesis was assessed by tube formation using endothelial cells obtained from bovine thoracic aorta (BAECs) grown on a layer of collagen type I. Addition of PMNs to BAECs weakly induced angiogenesis. The angiogenesis induced by PMNs alone was further enhanced by treatment of the PMNs with N-formyl-methionyl-leucyl-phenylalanine (FMLP), a selective activator of PMN. The involvement of PMN adhesion to BAECs via adhesion molecules in angiogenesis was investigated by using monoclonal antibodies against E-selectin and intercellular adhesion molecule-1 (ICAM-1). These antibodies blocked both the PMN adhesion to BAECs and the enhancement of angiogenesis induced by FMLP-treated PMNs. Furthermore, the enhancement of angiogenesis by FMLP-treated PMNs was blocked by catalase, a scavenging enzyme of H2O2, but not by superoxide dismutase (SOD). These results suggest that PMNs induce angiogenesis in vitro, and that the mechanism of stimulation of angiogenesis by PMNs may involve the adherence of PMNs to endothelial cells via E-selectin and ICAM-1, and H2O2, but not superoxide. Thus, activated PMNs in pathological states may not only induce tissue injury, but may also function as regulators of angiogenesis.

BUFALIN INHIBITS ENDOTHELIAL CELL PROLIFERATION AND ANGIOGENESIS IN VITRO

We have investigated the effects of bufalin, one of the prominent components in Chinese toad venom, on proliferation of bovine aortic endothelial (BAE) cells and tube formation in three-dimensional type I collagen matrix. In the present study, bufalin potently inhibited the formation of capillary-like tubular networks in a dose-dependent manner. Bufalin also inhibited the proliferation of BAE cells at the same concentration (5 nM) that the tube formation was inhibited. As a potent inhibitor of endothelial cell proliferation, bufalin specifically prevented the entry of BAE cells into the G0/G1 phase of a cell cycle. These findings suggest that in vitro angioinhibitory action of bufalin may be induced by the proliferation inhibition of endothelial cells through the arrest at the G2/M phase of a cell cycle.

Angiogenesis Induced by Tissue Factor in Vitro and in Vivo

The purpose of this study was to investigate the effects of tissue factor (thromboplastin), the initiating factor of the extrinsic clotting system, on angiogenesis in vivo and in vitro. In vivo angiogenesis was examined using a diffusion chamber assay in rats. After a week of implantation of the diffusion chambers containing tissue factor (0.5 or 5.0 mg/ mL), angiogenesis was enhanced two to three times as compared with the control. In vitro, an addition of 30 microg/mL of tissue factor enhanced angiogenesis in bovine aorta endothelial cells, which were cultured in collagen type gel 2.3-fold as compared with the control, and the angiogenesis was inhibited by antitissue factor antibody. Furthermore, tissue factor (30 microg/mL)-induced angiogenesis in bovine aorta endothelial cells was inhibited by the addition of coagulation factors II, VII, and IX. These results suggest that tissue factor directly induce angiogenesis, independently of the coagulation pathway.

Two novel oleanolic acid saponins having a sialyl Lewis X mimetic structure from Achyranthes fauriei root

Two novel triterpene glycosides, achyranthosides E and F, were isolated as methyl esters from the root of Achyranthes fauriei, an antiinflammatory medicinal plant. Their structures were characterized as oleanolic acid glucuronides having unique substituents composed of C6H9O5 and C9H15O7, respectively, at the C-3 position of glucuronic acid. These compounds are active components which can inhibit the excess recruiting of neutrophiles to injured tissues 1,000 times more potently than sialyl Lewis X.

Modulation of actomyosin ATPase by goniodomin A differs in types of cardiac myosin

Goniodomin A causes the conformational change of actin to modify actomyosin ATPase activity [Furukawa, K.-I., Sakai, K., Watanabe, S., Maruyama, K., Murakami, M., Yamaguchi, K., Ohizumi, Y., 1993. Goniodomin A induces modulation of actomyosin ATPase activity mediated through conformational change of actin. J. Biol. Chem. 268, 26026-26031]. Goniodomin A inhibited the ATPase activities of atrial myofibrils, myosin B and reconstituted actomyosin in a concentration-dependent manner. Interestingly, these ATPase activities of ventricular muscle were enhanced by goniodomin A (3 x 10(-8)-3 x 10(-7) M), but were decreased when the concentration was further raised. The stimulatory effect of goniodomin A was significantly inhibited by troponin tropomyosin complex. These results suggest that goniodomin A affects actin to modify cardiac actomyosin ATPase activity, and that this modulation differs in types of cardiac myosin.

UV-B and lipid hydroperoxides promote conjunctival epithelial cell migration

Abstract The idea that sunlight itself may be a major risk factor in certain eye diseases has been widely accepted. Peroxides or radicals arising from UV irradiation may induce factors leading to cell migration. To clarify this relationship further, we have examined the effects of UV radiation and lipid hydroperoxides on cell migration of conjunctival epithelial cells in culture from palpebral, fornical and bulbar regions. The cell migration from each region was enhanced by UV-B irradiation at 50 mJ/cm 2 and linoleic acid hydroperoxide (LHP) at 10 −7 M concentration. In contrast, cell migration was suppressed by UV-B irradiation at 100 mJ/cm 2 and LHP at 10 −5 M. Both UV-B and LHP induced cell migration was also suppressed by a vitamin E and C conjugated antioxidant (EPC-K1) which has radical scavenging activity. These data suggest that reactive oxygen intermediates play a prominent role in cell migration.

Angiogenesis induced by adhesion between polymorphonuclear leukocyte and endothelial cell via intercellular adhesion molecule-1

We demonstrate that adhesion between polymorphonuclear leukocyte (PMN) and endothelial cells (ECs) is concerned with induction of angiogenesis of ECs. For the tube formation assay, ECs obtaine from bovine thoracic aorta (BAECs) grown on a layer of collagen type I were used. Addition of PMNs treated with N-formyl-methionyl-leucy phenylalanine (FMLP), a selective activator of PMN induced angiogenesis The angiogenesis was blocked by monoclonal antibodies against E-selectin and intercellular adhesion molecule-1 (ICAM-1) which inhibit the adhesion between PMN and EC. Ets-1, which stimulates metalloproteinase gene transcription or integrin s3, has a key role in angiogenesis. Addition of activate PMNs to ECs stimulated the angiogenesis and Ets-1 expression. Both the angiogenesis and the Ets-1 expression induced by PMNs were reduced by ets-1 antisense oligonucleotide. On the other hand, PMN-induced Ets-1 expression was reduced by a monoclonal antibody against ICAM-1 but not E-selectin despite the inhibition of PMN-induced angiogenesis by both antibodies. The enhancement of angiogenesis by FMLP-treated PMNs Was blocked by catalase, a scavenging enzyme of H2O2, but not by superoxide dismutase (SOD). Interestingly, the stimulation of angiogenesis by H2O2 without PMNs was inhibited by anti E-selectin antibody but not anti ICA1V 1. ICAM-1 stimulation occurred by ICAM-1 cross-linking enhanced angio genesis. These findings indicated that PMN adhesion was related with the induction of angiogenesis, and ICAM-1 in endothelial cells acted as a signaing receptor to induce Ets-1 expression, whereas E-selectin seemed to function in the formation of tube-like structures in vascular endothelial cell cultures.