• Journal of Life Science
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• v.25 no.4
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• pp.416-424
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• 2015

Pinosylvin is a stilbenoid found in the Pinus species. Pinosylvin at ~pM to ~nM concentrations induces cell proliferation, cell migration and anti-inflammatory activity in endothelial cells. However, it was recently reported that pinosylvin at high concentrations (50 to 100 μM) induces cell death in bovine aortic endothelial cells. In this study, we conducted a series of experiments to discover how pinosylvin at a high concentration (50 μM) induces endothelial cell death. Pinosylvin at the high concentration was shown to induce endothelial cell apoptosis through enhancing caspase-3 activity, flip-flop of phosphatidyl serine, and nuclear fragmentation. We found that pinosylvin at the high concentration additively increased caspase-3 activity enhanced by serum-starvation or treatment with 100 μM etoposide. We also determined that pinosylvin at the high concentration promoted activations of c-Jun N-terminal kinase (JNK) and endothelial nitric oxide synthetase (eNOS). We further ran a series of experiments to find out which signaling molecule plays a critical role in the pinosylvin-induced apoptosis. We finally found that SP-600125, a JNK inhibitor, had an inhibitory effect on the pinosylvin-induced endothelial cell death, but L-NAME, an eNOS inhibitor, had no effect. These data indicate that JNK is involved in the pinosylvin-induced apoptosis. Collectively, pinosylvin at high doses induces cell apoptosis via JNK activation.

• BMB Reports
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• v.42 no.9
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• pp.561-567
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• 2009

Although many plant-derived phenolic compounds display antioxidant effects in biological systems, their mechanism of action remains controversial. In this study, the mechanism by which p-coumaric acid (p-CA) performs its antioxidant action was investigated in bovine aortic endothelial cells under oxidative stress due to high levels of glucose (HG) and arachidonic acid (AA), a free fatty acid. p-CA prevented lipid peroxidation and cell death due to HG+AA without affecting the production of reactive oxygen species. The antioxidant effect of p-CA was not decreased by buthionine-(S,R)-sulfoximine, an inhibitor of cellular GSH synthesis. In contrast, pretreatment with p-CA caused the induction of peroxidases that decomposed t-butyl hydroperoxide in a p-CA-dependent manner. Furthermore, the antioxidant effect of p-CA was significantly mitigated by methimazole, which was shown to inhibit the catalytic activity of 'p-CA peroxidases' in vitro. Therefore, it is suggested that the induction of these previously unidentified 'p-CA peroxidases' is responsible for the antioxidant effect of p-CA.

• Journal of Life Science
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• v.20 no.6
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• pp.914-921
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• 2010

In this study, we carried out a series of experiments to know whether melatonin, an anti-oxidative and immunosuppressive agent, played an important role in endothelial cells. It was revealed that melatonin had little or no effect on endothelial proliferation, cell death or migration. Additionally, melatonin had no effect on adhesion of THP-1 leukocytes to bovine aortic endothelial cells (BAECs) and THP-1 homotypic cell aggregation. In contrast, it was shown that melatonin diminished the basal level of nitric oxide by PP2A-mediated dephosphorylation of endothelial nitric oxide synthase (eNOS), leading to enhanced detachment of BAEC from the extracellular matrix. Collectively, melatonin in high doses decreases the NO production via regulations of PP2A and eNOS activities, inducing detachment of endothelial cells, a possible initial step for thrombosis.

• The Journal of Korean Medicine
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• v.24 no.4
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• pp.71-81
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• 2003

So-Hap-Hyang-Won, a traditional oriental medicine used in the treatment of stroke patients, was examined for its ability to reverse the cell damage caused by lipid peroxidation products and oxidative stress in bovine aortic endothelial cells (BAEC). The effects of herbal medicine on cell proliferation and recovery of oxidative damaged situation were studied in BAEC, which was considered an appropriate in vitro model for stroke resulting from various vascular diseases prevalent in advanced age. In a clinical study of stroke patients, So-Hap-Hyang-Won appeared to improve considerably arm and leg movements as well as consciousness disturbance condition, compared with other traditional medicines used for stroke. When BAEC were treated with extracts of the lyophilized herbal medicines, only that of So-Hap-Hyang-Won stimulated cell proliferation and showed no toxicity even at high concentrations. In studies of BAEC treated with extracts of the lyophilized material of the 14 components of So-Hap-Hyang-Won, only the extract of Foeniculi Fructus stimulated cell growth at all concentrations tested. Moreover, when cells were treated with Foeniculi Fructus (10 and 100 mg/ml) extract after prior exposure to t-BHP ($l0\mu\textrm{M}$) or HNE ($0.2\mu\textrm{M}$), lipid peroxidation products which are known to be involved in aging and vascular diseases, or after the exposure to SIN-l ($500\mu\textrm{M}$), which generates nitric oxide (NO) and other reactive oxygen species, there was substantial recovery from the oxidative damage, presumably due to the radical-scavenging effect of Foeniculi Fructus extract. Foeniculi Fructus not only showed stimulatory effects on cell growth and cell damage repair in BAEC, but also appeared to show the most anti-aging activity among all the herbal components of So-Hap-Hyang-Won.

• Biomolecules & Therapeutics
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• v.28 no.6
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• pp.549-560
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• 2020

Although DNA damage responses (DDRs) are reported to be involved in nitric oxide (NO) production in response to genotoxic stresses, the precise mechanism of DDR-mediated NO production has not been fully understood. Using a genotoxic agent aphidicolin, we investigated how DDRs regulate NO production in bovine aortic endothelial cells. Prolonged (over 24 h) treatment with aphidicolin increased NO production and endothelial NO synthase (eNOS) protein expression, which was accompanied by increased eNOS dimer/monomer ratio, tetrahydrobiopterin levels, and eNOS mRNA expression. A promoter assay using 5'-serially deleted eNOS promoters revealed that Tax-responsive element site, located at -962 to -873 of the eNOS promoter, was responsible for aphidicolin-stimulated eNOS gene expression. Aphidicolin increased CREB activity and ectopic expression of dominant-negative inhibitor of CREB, A-CREB, repressed the stimulatory effects of aphidicolin on eNOS gene expression and its promoter activity. Co-treatment with LY294002 decreased the aphidicolin-stimulated increase in p-CREB-Ser¹³³ level, eNOS expression, and NO production. Furthermore, ectopic expression of dominant-negative Akt construct attenuated aphidicolin-stimulated NO production. Aphidicolin increased p-ATM-Ser¹⁹⁸¹ and the knockdown of ATM using siRNA attenuated all stimulatory effects of aphidicolin on p-Akt-Ser⁴⁷³, p-CREB-Ser¹³³, eNOS expression, and NO production. Additionally, these stimulatory effects of aphidicolin were similarly observed in human umbilical vein endothelial cells. Lastly, aphidicolin increased acetylcholine-induced vessel relaxation in rat aortas, which was accompanied by increased p-ATM-Ser¹⁹⁸¹, p-Akt-Ser⁴⁷³, p-CREB-Ser¹³³, and eNOS expression. In conclusion, our results demonstrate that in response to aphidicolin, activation of ATM/Akt/CREB/eNOS signaling cascade mediates increase of NO production and vessel relaxation in endothelial cells and rat aortas.

• Archives of Pharmacal Research
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• v.20 no.6
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• pp.539-544
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• 1997

Anti-metastatic activities of IH901, an intestinal bacterial metabolic derivative formed from Ginseng protopanaxadiol saponins, was determined in vitro and in vivo. Under in vitro conditions, IH901 inhibited the migration of bovine aortic endothelial cells 25 times stronger than suramin and suppressed the invasion of HT1080 human fibrosarcoma cells into reconstituted basement membrane components of Matrigel 1000 times stronger than RGDS peptide. IH901 also showed inhibitory effect on type-IV collagenase secretion from HT 1080 cells and platelet aggregation. When the anti-metastatic activity of IH901 was evaluated in comparison with that of 5-FU using a spontaneous lung metastatic model of Lewis lung carcinoma, the administration of IH901 (10 mg/kg p. o.) to tumor-bearing mice led to a significant decrease in lung metastasis (43% of untreated control), which was slightly more effective than that obtained with 5-FU (56% of control). Thus, IH901 seems to exhibit its anti-metastatic activity partly through the inhibition of tumor invasion which results from the blockade of type IV collagenase secretion and also through anti-platelet and anti-angiogenic activities.

• Biomolecules & Therapeutics
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• v.22 no.6
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• pp.510-518
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• 2014

Chronic (>24 h) exposure of arsenite, an environmental toxicant, has shown the decreased nitric oxide (NO) production in endothelial cells (EC) by decreasing endothelial NO synthase (eNOS) expression and/or its phosphorylation at serine 1179 ($eNOS-Ser^{1179}$ in bovine sequence), which is associated with increased risk of vascular diseases. Here, we investigated the acute (<24 h) effect of arsenite on NO production using bovine aortic EC (BAEC). Arsenite acutely increased the phosphorylation of $eNOS-Thr^{497}$, but not of $eNOS-Ser^{116}$ or $eNOS-Ser^{1179}$, which was accompanied by decreased NO production. The level of eNOS expression was unaltered under this condition. Treatment with arsenite also induced reactive oxygen species (ROS) production, and pretreatment with a ROS scavenger N-acetyl-L-cysteine (NAC) completely reversed the observed effect of arsenite on $eNOS-Thr^{497}$ phosphorylation. Although protein kinase C (PKC) and protein phosphatase 1 (PP1) were reported to be involved in $eNOS-Thr^{497}$ phosphorylation, treatment with PKC inhibitor, Ro318425, and overexpression of various PKC isoforms did not affect the arsenite-stimulated $eNOS-Thr^{497}$ phosphorylation. In contrast, treatment with PP1 inhibitor, calyculin A, mimicked the observed effect of arsenite on $eNOS-Thr^{497}$ phosphorylation. Lastly, we found decreased cellular PP1 activity in arsenite-treated cells, which was reversed by NAC. Overall, our study demonstrates firstly that arsenite acutely decreases NO production at least in part by increasing $eNOS-Thr^{497}$ phosphorylation via ROS-PP1 signaling pathway, which provide the molecular mechanism underlying arsenite-induced increase in vascular disease.

• Journal of Physiology & Pathology in Korean Medicine
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• v.16 no.4
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• pp.680-683
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• 2002

Bovine aortic endothelial cells(BAEC) produce two variant forms of laminin with a subunit composition of AB1B2 and A'B1B2. Analyses of the intracellular assembly of these subunits revealed that the B1B2 dimer formed first, and that A or A' joined to form the AB1B2 or A'B1B2 trimer. Angiostatic steroids shifted the relative size of the A and A' monomer pool in BAEC, and competition between the A and A' subunits in joining the B1B2 dimer produced AB1B2 and A'B1B2 in different ratios. This result suggests that subunit replacement is the general mechanism for producing laminin variants by various cells for tissue morphogenesis. When laminin subunits in BAEC were cross-linked with dithio-bis-succinimidylpropionate(DSP) and immunoprecipitated with anti-Iaminin antiserum, monomeric A,A',B1 and B2 monomers and the B1B2 dimer migrated as extremely large molecules in sodium dodecyl sulfate gel electrophoresis under nonreducing conditions. When the crosslinking disulfide bonds were cleaved under reducing conditions, they migrated as the usual subunits. This result suggests that molecular chaperones were involved in the process of the assembly and replacement of laminin subunits.

• THE JOURNAL OF KOREAN ORIENTAL ONCOLOGY
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• v.7 no.1
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• pp.61-75
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• 2001

The purpose of this study is to prove the angiogenesis effects of WhaYoungJiTongTang (hereinafter referred to as the 'WYJTT'). For the study, by utilizing liver cancer cell line; SK-HEP-1, lung cancer cell line: A549, stomach cancer cell line: AGS and bovine capillary endothelial cell: BCE, the effects of the WYJTT on toxicity and proliferation ability of cells and the effects on anti-angiogenesis of bovine capillary endothelial cell and of mice's aorta were studied. 1. Cell viability assay In comparison with the control group, when $100{\mu}g/ml$ of WYJTT was injected, the viability was reduced in SK-Hep-1, $400{\mu}g/ml$ in A549 and $200{\mu}g/ml$ in AGS. 2. Cell proliferation assay In comparison with the control group, when $600{\mu}g/ml$ of WYITT was injected, DNA synthesis was reduced to 35.1% in the SK-Hep-1, 56.0% in A549, and 25.8% in BCE (bovine capillary endothelial cell); and when $400{\mu}g/ml$ was injected, DNA synthesis was reduced to 12.1 in AGS. 3. Tube formation assay In the event that BCE is injected with WYJTT in each of its content gradient, the anti-angiogenesis was effective in amounts of $400{\mu}g/ml$ with 6 hours of the treatment. 4. Aortic ring assay In comparison with the control group, the angiogenesis was restricted to the remarkable degree in amount of $200{\mu}g/ml$: 10% in $400{\mu}g/ml$; and fully inhibited in each of $800{\mu}g/ml$ and $1600{\mu}g/ml$. As a result of the experiments mentioned above, WYJTT showed its anti-angiogenesis effects against cancer cell line.

• Macromolecular Research
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• v.8 no.6
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• pp.276-284
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• 2000

A wettability chemogradient on poly(L-lactide-co-glycolide) (PLGA) films was prepared by treating the films in air with corona from a knife-type electrode whose power increases gradually along the sample length. The PLGA surfaces oxidized gradually with the increasing corona power, and the wettability chemogradient was created on the surfaces as evidenced by the measurement of water contact angles and electron spectroscopy for chemical analysis. The wettability chemogradient PLGA surfaces were used to investigate the interaction of four different types of cells such as hepatoma (Hep G2), osteoblast (MG 63), bovine aortic endothelial (CPAE), and fibroblast (NIH/3T3) cells in terms of the surface hydrophilicity/hydrophobicity of PLGA. The cells adhered and grown on the chemogradient surface along the sample length were counted and observed by scanning electron microscopy. It was observed that the cells were adhered, spread, and grown more onto the positions with moderate hydrophilicity of the wettability chemogradient PLGA surface than the more hydrophobic or hydrophillic positions, regardless of the cell types used. The maximum adhesion and growth of the cells appeared at around water contact angles of 53～55°. This result seems closely related with the serum protein adsorption on the surface; the serum proteins were also adsorbed more onto the positions with moderate hydrophilicity of the wettability chemogradient surface. It seems that the wettability plays important roles for cell adhesion, spreading and growth on the PLGA surface. The surface modification technique used in this study may be applicable tothe area of tissue engineering for the improvement of tissue compatibility of films- or scaffold-type substrates.