ABSTRACT
Recent studies have shown that L-carnitine supplementation of sows during pregnancy and lactation enhances their reproductive performance, but the underlying mechanisms are still needed to be further confirmed. This study was conducted to investigate the function of L-carnitine on placental development, milk nutrient content and release of hormones in sows. In this experiment, 40 multiparous crossbred sows (Yorkshire × Landrace) were allotted to two groups fed diets with or without a supplemental 50 mg/kg L-carnitine. The experimental diets were fed from d 1 post-coitus until d 21 post-partum. L-carnitine-treated sow had fewer weak piglets (p < 0.05) and a greater percentage of oestrus by 5 after 5-d post-partum (p < 0.05) than control sows. The percentage fat from colostrum was greater in L-carnitine-treated sow than control sows (p < 0.05). L-carnitine-treated sows had greater plasma concentrations of triglyceride and insulin-like growth factor (IGF)-1 and lesser plasma concentrations of glucose and IGF-binding protein (IGFBP-3) on day 60 of pregnancy (p < 0.05). A clearer structure of chorions, better-developed capillaries and absence of necrosis were observed in L-carnitine-treated sows compared with control sows. The protein abundance of IGF-1 and IGF-2 in placental chorions was greater in L-carnitine-treated sows compared with control sows (p < 0.05). This study suggests that sows fed an L-carnitine supplemented diet during pregnancy improved reproductive performance through enhancement of placental development and by increasing IGF concentrations in blood plasma and placental chorions.
Subject(s)
Carnitine/metabolism , Chorion/drug effects , Insulin-Like Growth Factor I/metabolism , Milk/chemistry , Placentation/drug effects , Reproduction/drug effects , Swine/physiology , Animal Feed/analysis , Animal Nutritional Physiological Phenomena/drug effects , Animals , Carnitine/administration & dosage , Chorion/chemistry , Diet/veterinary , Dietary Supplements/analysis , Female , Milk/drug effects , PregnancyABSTRACT
STUDY QUESTION: Does coumestrol inhibit proliferation of human placental choriocarcinoma cells? SUMMARY ANSWER: Coumestrol promotes cell death in the choriocarcinoma cells by regulating ERK1/2 MAPK and JNK MAPK signaling pathways and through disruption of Ca2+ and ROS homeostasis. WHAT IS KNOWN ALREADY: A number of patients who suffer from choriocarcinomas fail to survive due to delayed diagnosis or a recurrent tumor and resistance to traditional chemotherapy using platinum-based agents and methotrexate. To overcome these limitations, it is important to discover novel compounds which have no adverse effects yet can inhibit the expression of a target molecule to develop, as a novel therapeutic for prevention and/or treatment of choriocarcinomas. STUDY DESIGN, SIZE, DURATION: Effects of coumestrol on human placental choriocarcinoma cell lines, JAR and JEG3, were assessed in diverse assays in a dose- and time-dependent manner. PARTICIPCANTS/MATERIALS, SETTING, METHODS: Effects of coumestrol on cell proliferation, apoptosis (annexin V expression, propidium iodide staining, TUNEL and invasion assays), mitochondria-mediated apoptosis, production of reactive oxygen species (ROS), lipid peroxidation, glutathione levels and endoplasmic reticulum (ER) stress proteins in JAR and JEG3 cells were determined. Signal transduction pathways in JAR and JEG3 cells in response to coumestrol were determined by western blot analyses. MAIN RESULTS AND THE ROLE OF CHANCE: Results of the present study indicated that coumestrol suppressed proliferation and increased apoptosis in JAR and JEG3 cells by inducing pro-apoptotic proteins, Bax and Bak. In addition, coumestrol increased ROS production, as well as lipid peroxidation and glutathione levels in JAR and JEG3 cells. Moreover, coumestrol-induced depolarization of mitochondrial membrane potential (MMP) and increased cytosolic and mitochondrial Ca2+ levels in JAR and JEG3 cells. Consistent with those results, treatment of JAR and JEG3 cells with a Ca2+ chelator and an inhibitor of IP3 receptor decreased coumestrol-induced depolarization of MMP and increased proliferation in JAR and JEG3 cells. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: A lack of in vivo animal studies is a major limitation of this research. The effectiveness of coumestrol to induce apoptosis of human placental choriocarcinoma cells requires further investigation. WIDER IMPLICATIONS OF THE FINDINGS: Our results indicate that coumestrol induces apoptotic effects on placental choriocarcinoma cells by regulating cell signaling and mitochondrial-mediated functions, with a potential to impair progression of the cancer. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by grants from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (No. HI15C0810 awarded to G.S. and HI17C0929 awarded to W.L.).
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Coumestrol/pharmacology , Epithelial Cells/drug effects , Gene Expression Regulation, Neoplastic , Mitochondria/drug effects , Phytoestrogens/pharmacology , Apoptosis/genetics , Calcium/agonists , Calcium/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Chorion/drug effects , Chorion/metabolism , Chorion/pathology , Dose-Response Relationship, Drug , Endoplasmic Reticulum Stress/drug effects , Epithelial Cells/metabolism , Epithelial Cells/pathology , Glutathione/metabolism , Humans , Lipid Peroxidation , MAP Kinase Kinase 4/genetics , MAP Kinase Kinase 4/metabolism , Membrane Potential, Mitochondrial/drug effects , Mitochondria/metabolism , Mitochondria/pathology , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Reactive Oxygen Species/agonists , Reactive Oxygen Species/metabolism , Signal Transduction , bcl-2 Homologous Antagonist-Killer Protein/agonists , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/agonists , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
Angiogenesis, which is required for physiological events, plays a crucial role in several pathological conditions, such as tumor growth and metastasis. The use of plant extracts is a cost effective and eco-friendly way to synthesize nanoparticles. In the present study, we investigated the anti-angiogenesis properties of silver nanoparticles synthesized using Saliva officinalis extract on chick chorioalantoic membrane. The production of nanoparticles was confirmed by the color change from yellow to brown observed after approximately 3 h at 37 °C. Then, the nanoparticles were characterized by UV-visible spectroscopy, FTIR, and TEM. The UV-visible spectroscopy results showed that the surface plasmon resonance band for AgNPs was around 430 nm. The intensity of the AgNP-specific absorption peak improved with an increase of 0.5 mL of extract into 10 mL of AgNO3 (2.5 mM). The FTIR results showed good interaction between the plant extracts and AgNPs. The TEM images of the samples revealed that the NPs varied in morphology and size from 1 to 40 nm; the average was recorded at 16.5 ± 1.2 nm. Forty Ross fertilized eggs were divided into four groups; the control and three experimental groups. On the 8th day, gelatin sponges containing albumin were placed on the chorioalantoic membrane and soaked with different concentrations of NPs. On the 12th day, all the cases were photographed using a photostereomicroscope. The number and the lengths of the vessels were measured using Image J software. The crown rump (CR) and weight of the embryo were also recorded. Then the hemoglobin content was measured using Drabkin's reagent kit for quantification of the blood vessel formation. According to the data analysis, the number and length of the blood vessels, as well as the CR and weight of the embryos reduced significantly compared to the control (p < 0.05), dose dependently. The total hemoglobin was quantified as an indicator of the blood vessel formation. The hemoglobin content in the treated samples with AgNPs decreased, which showed its inhibitory effect on angiogenesis.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Chorion/drug effects , Metal Nanoparticles , Plant Extracts/pharmacology , Salvia officinalis/chemistry , Silver/chemistry , Allantoin , Animals , Chick Embryo , Spectrophotometry, UltravioletABSTRACT
Control of vascular resistance and blood flow in the fetoplacental circulation is incompletely understood. Reactive oxygen species (ROS), physiological and pathophysiological regulators of vascular tone, are elevated in preeclampsia (PE), a disease of pregnancy characterized by increased fetoplacental vascular resistance. We tested the hypothesis that ROS modulate vascular reactivity in placental chorionic plate arteries. Wire myography was used to examine (1) the effects of acute exposure to ROS on arterial function in normal pregnancy and (2) the effects of maternal antioxidant supplementation on arterial reactivity in women at high risk for PE participating in the Vitamins in Pre-eclampsia (VIP) trial. ROS generated by xanthine plus xanthine oxidase enhanced basal tension, vasoconstriction in response to the thromboxane mimetic U46619, and relaxation in response to sodium nitroprusside. Hydrogen peroxide and peroxynitrite increased basal tone and relaxed preconstricted arteries (U44619), respectively. In women at risk for PE, chorionic plate artery constriction in response to U46619 was greater in the women receiving placebo compared to the women supplemented with the antioxidant vitamins C and E. ROS may regulate fetoplacental vascular resistance and blood flow in the short term, and chronic exposure to raised ROS could contribute to elevated fetoplacental vascular resistance in PE and fetal growth restriction (FGR).
Subject(s)
Chorion/physiology , Reactive Oxygen Species/pharmacology , Umbilical Arteries/physiology , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology , Adult , Antioxidants/pharmacology , Chorion/blood supply , Chorion/drug effects , Female , Humans , In Vitro Techniques , Myography , Placenta , Placental Circulation/drug effects , Pre-Eclampsia/physiopathology , Pregnancy , Regional Blood Flow , Umbilical Arteries/drug effects , Vasoconstriction/drug effects , Vitamins/pharmacology , Xanthine/pharmacology , Xanthine Oxidase/pharmacology , Young AdultABSTRACT
Previous studies have shown that supplementation of sow diets with L-carnitine increases the body weight of piglets at birth. This study was conducted to elucidate the reasons for this phenomenon. Three experiments with 24 (experiment 1), 40 (experiment 2) and 12 (experiment 3) sows were conducted. In all three experiments, sows were allotted to two groups which had free access to a nutritionally adequate diet. Sows of one group were supplemented with 125 mg L-carnitine/day during pregnancy; sows of the other group (control group) did not receive L-carnitine. In experiment 1, plasma samples were collected at day 95 of pregnancy, in experiment 2 plasma samples were collected at days 80 and 100 of pregnancy. In experiment 3, chorions of the sows were collected at parturition. L-carnitine-treated sows had higher plasma concentrations of total L-carnitine than control sows (p < 0.05). The number of piglets born and weights of litter and individual piglets at birth were not different between both groups in all three experiments. L-carnitine-treated sows had higher plasma concentrations of insulin-like growth factor-I (IGF-I) on day 80 of pregnancy (experiment 2, p < 0.05) and on day 95 (experiment 1, p < 0.10), and a higher plasma concentration of IGF-II on day 80 (experiment 2, p < 0.05) than control sows. Moreover, sows supplemented with L-carnitine had heavier chorions (+22%, p =0.10) with greater amounts of protein (+45%, p < 0.05) and DNA (+38%, p < 0.10) and a higher protein concentration of glucose transporter-1 (+62%, p < 0.05). Plasma concentrations of 17beta-oestradiol, progesterone and thyroid hormones as well as concentrations of urea and total free amino acids were not different between both groups of sows. Plasma concentrations of non-esterified fatty acids, ketone bodies, triacylglycerols and cholesterol were also largely indifferent between both groups of sows. In conclusion, this study shows that L-carnitine has less influence on lipid metabolism and utilization of nitrogen in pregnant sows but increases their plasma concentrations of IGFs. This in turn may enhance development of the placentae and the intrauterine nutrition of the fetuses. This may be the reason for increased birth weights observed in recent studies in sows supplemented with L-carnitine.
Subject(s)
Animal Nutritional Physiological Phenomena , Carnitine/pharmacology , Nutritional Requirements , Pregnancy, Animal/blood , Somatomedins/metabolism , Swine/physiology , Animals , Birth Weight/drug effects , Birth Weight/physiology , Carnitine/administration & dosage , Chorion/drug effects , Chorion/metabolism , Dose-Response Relationship, Drug , Female , Lipid Metabolism/drug effects , Litter Size/drug effects , Litter Size/physiology , Nitrogen/metabolism , Placenta/drug effects , Placentation , Pregnancy , Swine/bloodABSTRACT
Rigorous and systematic pre-clinical studies are necessary and essential to establish the efficacy and safety of Oriental herbs and formulas in order to transform traditional herbal practices into evidence-based medicine. Here we evaluated the anti-cancer activities of the ethanol extract of Ka-mi-kae-kyuk-tang (KMKKT), a formula of ten Oriental herbs, with a battery of in vitro and in vivo mechanism-based biomarkers involving angiogenesis, apoptosis and metastasis. The results show that KMKKT suppressed the vascular endothelial responses by inhibiting basic fibroblast growth factor (bFGF)-induced ERK1/2 phosphorylation, cell migration as well as tube formation in the human umbilical vein endothelial cell model, and decreased the hypoxia-induced HIF1alpha and vascular epithelial growth factor (VEGF) expression in the mouse Lewis lung carcinoma (LLC) cells in vitro, and inhibited the bFGF-induced angiogenesis in chick chorioallantoic membrane model, and in the Matrigel plugs in mice. Intraperitoneal delivery of KMKKT potently inhibited the growth of the subcutaneously inoculated LLC cells in syngenic mice. In addition, KMKKT inhibited the invasion ability of the mouse colon 26-L5 cancer cells in vitro and decreased their formation of liver metastasis when intraportally inoculated in syngenic mice. Furthermore, KMKKT suppressed the growth of the human PC-3 prostate cancer xenografts in athymic nude mice and averted the cancer-related body weight loss. The in vivo cancer growth suppression was associated with a decreased microvessel density and VEGF abundance as well as an increased PARP cleavage and the TUNEL-positive apoptosis. Together, our data support broad-spectra in vivo anti-cancer activities of KMKKT targeting angiogenesis, apoptosis and metastasis without any adverse effect on the body weight. This formula merits serious consideration for further evaluation for the chemoprevention and treatment of cancers of multiple organ sites.
Subject(s)
Apoptosis/drug effects , Neoplasm Metastasis/prevention & control , Neovascularization, Pathologic/prevention & control , Plant Extracts/pharmacology , Allantois/drug effects , Animals , Cell Division/drug effects , Cell Movement/drug effects , Cell Survival/drug effects , Cells, Cultured , Chick Embryo , Chorion/drug effects , Colonic Neoplasms/pathology , Disease Models, Animal , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Humans , Korea , Liver Neoplasms/pathology , Medicine, East Asian Traditional , Mice , Mice, Inbred BALB C , Umbilical VeinsABSTRACT
The tenebrioid beetle Palembus ocularis Casey 1891 has been used in the traditional medicine of Central and South America to treat Asthma bronchiale. Palembus ocularis has large pygidial glands filled with defence chemicals. The defence fluid was analysed by GLC and GLC-MS. The main components are quinones, such as hydroquinone, 2-ethylhydroquinone as major and 2-methylhydroquinone as minor metabolite. Furthermore, terpenes, fatty acids and their esters, cholesterol and lactones were discovered, with 1-pentadecene as a major component. Pharmacological experiments were carried out with the defence fluid and isolated compounds in order to find out if a rational base exists for the use in traditional medicine. 5-Lipoxygenase is inhibited by crude extracts and hydroquinone. Moreover, a combination of different hydroquinones was 20-times more effective than individual substances suggesting a synergistic effect. The inhibitory effect could be further increased with addition of 1-pentadecene. Anti-inflammatory activity was determined by HET-CAM. The polar extracts of Palembus ocularis showed anti-inflammatory activity in this system. Pure hydroquinone exhibited a similar activity as crude extracts indicating that it constitutes the active principle. The 5-LOX and HET-CAM-assays provide good evidence that hydroquinones in the defence fluid of Palembus ocularis have anti-inflammatory properties which would explain the traditional use of this beetle to treat asthma.
Subject(s)
Anti-Inflammatory Agents/therapeutic use , Coleoptera/metabolism , Hydroquinones/therapeutic use , Inflammation/drug therapy , Animals , Arachidonate 5-Lipoxygenase/metabolism , Blood Vessels/drug effects , Chick Embryo , Chorion/drug effects , Erythrocytes/drug effects , Gas Chromatography-Mass Spectrometry , Humans , Neutrophils/drug effectsABSTRACT
Chorioamniotic infection is a leading cause of preterm premature rupture of fetal membranes (amnion and chorion). Bacterial infection induces an inflammatory response characterized by elevated production of proinflammatory cytokines; the latter activate the production of both PGs that stimulate uterine contractions, and matrix metalloproteinases (MMPs) that degrade the extracellular matrix of the chorioamniotic membranes. The inflammatory response is under the control of cAMP content, which is partly regulated by phosphodiesterases (PDE). In this study, we investigated the role of the PDE4 family in the inflammatory process triggered by LPS in a model of amniochorionic explants. We found that PDE4 family is the major cAMP-PDE expressed in human fetal membranes and that PDE4 activity is increased by LPS treatment. Selective inhibition of PDE4 activity affected LPS signaling, because PDE4 inhibitors (rolipram and/or cilomilast) reduced the release of the proinflammatory cytokine TNF-alpha and increased the release of the anti-inflammatory cytokine IL-10. PDE4 inhibition reduced cyclooxygenase-2 protein expression and PGE(2) production and also modulated MMP-9, a key mediator of the membrane rupture process, by inhibiting pro-MMP-9 mRNA expression and pro-MMP-9 activity. These results demonstrate that the PDE4 family participates in the regulation of the inflammatory response associated with fetal membrane rupture during infection. The PDE4 family may be an appropriate pharmacological target for the management of infection-induced preterm delivery.
Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/physiology , Amnion/enzymology , Amnion/immunology , Chorion/enzymology , Chorion/immunology , Dinoprostone/biosynthesis , Lipopolysaccharides/immunology , Matrix Metalloproteinase 9/metabolism , 3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , Adjuvants, Immunologic/pharmacology , Adjuvants, Immunologic/physiology , Amnion/drug effects , Amnion/metabolism , Chorion/drug effects , Chorion/metabolism , Cyclic AMP/antagonists & inhibitors , Cyclic AMP/physiology , Cyclic Nucleotide Phosphodiesterases, Type 4 , Cyclooxygenase 2 , Enzyme Activation/drug effects , Enzyme Activation/physiology , Enzyme Precursors/metabolism , Female , Humans , Immune Sera/pharmacology , Interleukin-10/immunology , Interleukin-10/metabolism , Membrane Proteins , Phosphodiesterase Inhibitors/pharmacology , Pregnancy , Prostaglandin-Endoperoxide Synthases/biosynthesis , Rolipram/pharmacology , Tissue Culture Techniques , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolism , Up-Regulation/drug effects , Up-Regulation/immunologyABSTRACT
Linoleic acid (18:2n-6) is metabolised to arachidonic acid (20:4n-6), the precursor for 2-series prostaglandins (PGs). Increased consumption of 18:2n-6 during pregnancy may thus modify PG synthesis during labour. We have investigated whether increased 18:2n-6 composition during gestation altered the fatty acid consumption and PG synthesis of maternal and fetal tissues in the sheep. Ewes were fed a control diet or a diet providing 40% more 18:2n-6 from 96 days gestation. Half of each group received dexamethasone on day 136 to up-regulate the PG synthetic pathways promoting parturition. Maternal and fetal tissues were collected at 138 days. The 18:2n-6 diet significantly increased the 20:4n-6 content of maternal plasma, fetal plasma and allantochorion (51-81%) phosphatidylcholine, and fetal liver (40%) and maternal caruncular endometrium (57%) phosphatidylethanolamine. Increased 18:2n-6 intake increased production of PGF(2alpha) and PGE(2) in all placental tissues (maternal caruncular and intercaruncular endometrium and fetal allantochorion) by 23-98%, whereas dexamethasone increased it by 32-142%. This suggests that consumption of an 18:2n-6-enriched diet in late pregnancy enhanced placental PG production by increasing the supply of 20:4n-6. Variations in the extent to which the diet altered the polyunsaturated fatty acid (PUFA) content of the different tissues indicated complex interactions between nutrient availability and metabolic adaptation.
Subject(s)
Dinoprostone/biosynthesis , Fatty Acids/metabolism , Fetal Blood/metabolism , Linoleic Acid/administration & dosage , Placenta/metabolism , Pregnancy, Animal/blood , Allantois/drug effects , Allantois/metabolism , Animals , Chorion/drug effects , Chorion/metabolism , Dietary Supplements , Dinoprostone/analysis , Endometrium/drug effects , Endometrium/metabolism , Fatty Acids/blood , Fatty Acids/chemistry , Female , Fetal Blood/drug effects , Maternal-Fetal Exchange , Phosphatidylcholines/metabolism , Phosphatidylethanolamines/metabolism , Pregnancy , SheepABSTRACT
The Chinese folk medicine Shiraia bambusicola has long been utilized in the treatment of rheumatoid arthritis, a disease in which angiogenesis plays an important role. We report here the isolation of the compound Shiraiachrome A from S. bambusicola and the demonstration of its anti-angiogenic properties. We found that Shiraiachrome A significantly inhibited the proliferation, migration, and tube formation of human microvascular endothelial cells (HMEC) in a dose-dependent manner, with average IC(50) values of 2.1+/-0.36, 1.97+/-0.44, and 1.65+/-0.59 microM, respectively. In addition, Shiraiachrome A inhibited the formation of new microvessels in a rat aorta culture model as well as in the chick embryo chorioallantoic membrane (CAM) assay. Investigation of the mechanism of action of Shiraiachrome A demonstrated that this compound suppressed the autophosphorylation of four receptor tyrosine kinases (RTKs), with IC(50) values ranging from 2.2 to 4.3 microM. These results suggest that Shiraiachrome A inhibits angiogenesis by blocking growth factor-stimulated autophosphorylation of RTKs. These findings also indicate that Shiraiachrome A may be a potent therapeutic agent for angiogenesis-related diseases such as cancer, rheumatoid arthritis, and diabetic retinopathy.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Arthritis, Rheumatoid/drug therapy , Drugs, Chinese Herbal/pharmacology , Perylene/analogs & derivatives , Perylene/pharmacology , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/isolation & purification , Animals , Aorta, Thoracic/cytology , Aorta, Thoracic/drug effects , Capillaries/drug effects , Capillaries/ultrastructure , Cell Movement/drug effects , Cell Proliferation/drug effects , Chick Embryo , Chorion/drug effects , Dose-Response Relationship, Drug , Drugs, Chinese Herbal/chemistry , Endothelial Cells/drug effects , Immunoblotting , Indicators and Reagents , Mice , Microtubules/drug effects , Microtubules/ultrastructure , NIH 3T3 Cells , Perylene/chemistry , Perylene/isolation & purification , Phosphorylation , Rats , Rats, Sprague-Dawley , Receptor Protein-Tyrosine Kinases/metabolismABSTRACT
A homoisoflavanone, 5,7-dihydroxy-3-(3-hydroxy-4-methoxybenzyl)-6-methoxychroman-4-one ( 1), was isolated from the bulb of Cremastra appendiculata (D. Don) Makino (Orchidaceae) as a potent inhibitor of angiogenesis. It inhibited basic fibroblast growth factor (bFGF)-induced in vitro angiogenesis and in vivo angiogenesis of the chorioallantoic membrane (CAM) of chick embryo without showing any toxicity.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Fibroblast Growth Factor 2/drug effects , Orchidaceae , Phytotherapy , Plant Extracts/pharmacology , Angiogenesis Inhibitors/administration & dosage , Angiogenesis Inhibitors/therapeutic use , Animals , Chick Embryo , Chorion/blood supply , Chorion/drug effects , Isoflavones/administration & dosage , Isoflavones/pharmacology , Isoflavones/therapeutic use , Neovascularization, Physiologic/drug effects , Plant Extracts/administration & dosage , Plant Extracts/therapeutic useABSTRACT
8-Prenylnaringenin is a recently discovered phytoestrogen. Using an in vitro model of angiogenesis in which endothelial cells can be induced to invade a three-dimensional collagen gel within which they form capillary-like tubes, we demonstrate that 8-prenylnaringenin inhibits angiogenesis induced by basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), or the synergistic effect of the two cytokines in combination, with an IC(50) of between 3 and 10 microM. This effect was seen with bovine microvascular endothelial cells derived from the adrenal cortex (BME cells) and with endothelial cells from the bovine thoracic aorta (BAE cells). The inhibitory effects of 8-prenylnaringenin were found to be roughly equipotent to those of genistein that has previously been shown to inhibit angiogenesis in vitro. Early chorioallantoic membrane (CAM) assay results showed reductions in both vessel lengths and vein diameters, with similar potency in the 8-prenylnaringenin and genistein groups. Similar effects on the CAM vessels were seen when the two substances were co-added. These findings suggest that 8-prenylnaringenin has potential therapeutic applications for diseases in which angiogenesis is an important component.
Subject(s)
Endothelial Cells/drug effects , Flavanones/pharmacology , Isoflavones/pharmacology , Neovascularization, Physiologic/drug effects , Plant Preparations/pharmacology , Allantois/drug effects , Animals , Cells, Cultured , Chorion/drug effects , Fibroblast Growth Factor 2/pharmacology , Genistein/pharmacology , Growth Inhibitors/pharmacology , Humans , In Vitro Techniques , Phytoestrogens , Recombinant Proteins/pharmacology , Vascular Endothelial Growth Factor A/pharmacologyABSTRACT
The in vivo test on the chorioallantoic membrane of the fertilized hen's egg (CAM assay) is a current method to determine antiangiogenic, antiinflammatory activity and toxic effects of individual compounds or complex plant extracts. The method is used for testing natural compounds in small amounts for revealing various modes of action and the complex mechanisms related to angiogenesis and inflammation. Furthermore, possible side effects such as membrane irritation, toxic, and anticoagulant properties of the investigated material in question can be detected. For the evaluation, the essential oil obtained by hydrodistillation of the aerial parts of Origanum onites L., a common spice and medicinal plant, was tested for its effect in the chorioallantoic membrane (CAM) assay. The essential oil composition was revealed by means of gas chromatography-mass spectrometry (GC-MS). Eighty three components were identified, representing 99.1% of the total oil. Carvacrol, thymol, p-cymene, and gamma-terpinene were found as major components and were also individually tested in the CAM assay. Along with the monoterpenes carvacrol and thymol, their methyl ether derivatives were also examined for comparison of their physiological action. Neither the essential oil nor its components showed any pronounced antiinflammatory or antiangiogenic property in the CAM assay, at 10-250 microg/pellet. However, the irritant effect of the essential oil was linked to thymol in a dose-response fashion, up to 10 microg/pellet, where it was still showing irritation.
Subject(s)
Allantois/drug effects , Chorion/drug effects , Oils, Volatile/chemistry , Angiogenesis Inhibitors/analysis , Animals , Anti-Inflammatory Agents/analysis , Chickens , Cyclohexane Monoterpenes , Cymenes , Female , Gas Chromatography-Mass Spectrometry , Monoterpenes/analysis , Monoterpenes/pharmacology , Thymol/analysis , Thymol/pharmacologyABSTRACT
The anticarcinogenic properties of conjugated linoleic acid (CLA) are, at least partially, attributed to its ability to interrupt the n-6 polyunsaturated fatty acid (PUFA) metabolic pathway for the biosynthesis of eicosanoids, including prostaglandins (PG). Both PGE(2) and PGF(2alpha) play key roles in parturition. In the present study, we compared the effects of CLA (a mixture of cis- and trans-9, 11- and -10, 12-octadecadienoic acid) and linoleic acid (LA) on PG production by cells isolated from maternal intercotyledonary endometrium, fetal allantochorion and amnion from late pregnant ewes. The results demonstrated that supplementation of LA and CLA significantly affected both the proportions and the amounts of PGs produced by all three tissue types. The ability of the uterus and placenta to respond to oxytocin (OT, endometrium only) and lipopolysaccharide (LPS) was also affected. LA inhibited PGE(2) and PGF(2alpha) production in the absence or presence of either oxytocin or LPS. In endometrial cells with or without oxytocin or LPS, CLA dose-dependently suppressed PGF(2alpha) generation, whereas low doses of CLA (20 microM) increased PGE(2) generation. Supplementation with CLA therefore increased the PGE(2)/PGF(2alpha) ratio in the endometrial cells. These results suggest that dietary supplementation of LA or CLA may affect both the initiation and progression of parturition.
Subject(s)
Endometrium/drug effects , Endometrium/metabolism , Extraembryonic Membranes/drug effects , Extraembryonic Membranes/metabolism , Linoleic Acids/toxicity , Prostaglandins/biosynthesis , Allantois/drug effects , Allantois/metabolism , Amnion/drug effects , Amnion/metabolism , Animals , Chorion/drug effects , Chorion/metabolism , Dietary Supplements/toxicity , Dinoprost/biosynthesis , Dinoprostone/biosynthesis , Female , In Vitro Techniques , Parturition/drug effects , Parturition/metabolism , Pregnancy , SheepABSTRACT
Increased local osteoclast (OC)-mediated bone resorption coincides with angiogenesis in normal bone development and fracture repair, as well as in pathological disorders such as tumor-associated osteolysis and inflammatory-related rheumatoid arthritis or periodontal disease. Angiogenic stimulation causes recruitment, activation, adhesion, transmigration, and differentiation of hematopoietic cells which may therefore enable greater numbers of pre-OC to emigrate from the circulation and develop into bone-resorptive OCs. A chick chorioallantoic membrane (CAM) model, involving coimplantation of a stimulus in an agarose plug directly adjacent to a bone chip was used to investigate if a potent angiogenic stimulator, basic fibroblast growth factor (bFGF), could promote OC recruitment, differentiation, and resorption in vivo. Angiogenesis elicited by bFGF on the CAM was accompanied by increased OC formation and bone pit resorption (both overall and on a per OC basis) on the bone implants in vivo. In complementary in vitro assays, bFGF did not directly stimulate avian OC development from bone marrow mononuclear cell precursors, consistent with their low mRNA expression of the four avian signaling FGF receptors (FGFR)-1, FGFR-2, FGFR-3, and FGFR-like embryonic kinase (FREK). In contrast, bFGF activated isolated avian OC bone pit resorption via mechanisms inhibited by a selective cyclo-oxygenase (COX)-2 prostaglandin inhibitor (NS-398) or p42/p44 MAPK activation inhibitor (PD98059), consistent with a relatively high expression of FGFR-1 by differentiated avian OCs. Thus, bFGF may sensitively regulate local bone resorption and remodeling through direct and indirect mechanisms that promote angiogenesis and OC recruitment, formation, differentiation, and activated bone pit resorption. The potential for bFGF to coinduce angiogenesis and OC bone remodeling may find clinical applications in reconstructive surgery, fracture repair, or the treatment of avascular necrosis. Alternatively, inhibiting such bFGF-dependent processes may aid in the treatment of inflammatory-related or metastatic bone loss.
Subject(s)
Allantois/drug effects , Bone Resorption/chemically induced , Chorion/drug effects , Fibroblast Growth Factor 2/pharmacology , Neovascularization, Physiologic/drug effects , Osteoclasts/drug effects , Animals , Cell Differentiation/drug effects , Cells, Cultured/drug effects , Chick Embryo , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Cyclooxygenase Inhibitors/pharmacology , Drug Implants , Flavonoids/pharmacology , Gene Expression Regulation, Developmental/drug effects , Humans , Isoenzymes/antagonists & inhibitors , Membrane Proteins , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Nitrobenzenes/pharmacology , Prostaglandin-Endoperoxide Synthases , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptor Protein-Tyrosine Kinases/biosynthesis , Receptor Protein-Tyrosine Kinases/genetics , Receptor, Fibroblast Growth Factor, Type 4 , Receptors, Fibroblast Growth Factor/biosynthesis , Receptors, Fibroblast Growth Factor/genetics , Recombinant Proteins/pharmacology , Sulfonamides/pharmacologyABSTRACT
Propolis, obtained from honeybee hives, has been used in Oriental folk medicine as an anti-inflammatory, anti-carcinogenic, and immunomodulatory agent. There is considerable evidence suggesting that angiogenesis and chronic inflammation are codependent. Blockage of angiogenesis results in an anti-inflammatory effect. Ethanol (EEP) and ether extracts of propolis (REP), and caffeic acid phenethyl ester (CAPE), an active component of propolis, were examined for their anti-angiogenic activities using the chick embryo chorioallantoic membrane (CAM), and the calf pulmonary arterial endothelial (CPAE) cell proliferation, assays. The presence of EEP, REP and CAPE inhibited angiogenesis in the CAM assay and the proliferation of CPAE cells. The results suggest that anti-angiogenic activities of EEP, REP and CAPE are also responsible for their anti-inflammatory effect.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Phenylethyl Alcohol/analogs & derivatives , Propolis/pharmacology , Animals , Caffeic Acids/pharmacology , Cattle , Cell Division/drug effects , Chick Embryo , Chorion/drug effects , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Ethanol , Keratolytic Agents/pharmacology , Membranes/drug effects , Phenylethyl Alcohol/pharmacology , Pulmonary Artery/cytology , Pulmonary Artery/drug effects , Solvents , Tetrazolium Salts , Thiazoles , Tretinoin/pharmacologyABSTRACT
Conformationally altered proteins and protein fragments derived from the extracellular matrix and hemostatic system may function as naturally occurring angiogenesis inhibitors. One example of such a protein is cleaved high molecular weight kininogen (HKa). HKa inhibits angiogenesis by inducing apoptosis of proliferating endothelial cells, effects mediated largely by HKa domain 5. However, the mechanisms underlying the antiangiogenic activity of HKa have not been characterized, and its binding site on proliferating endothelial cells has not been defined. Here, we report that the induction of endothelial cell apoptosis by HKa, as well as the antiangiogenic activity of HKa in the chick chorioallantoic membrane, was inhibited completely by antitropomyosin monoclonal antibody TM-311. TM-311 also blocked the high-affinity Zn2+-dependent binding of HKa to both purified tropomyosin and proliferating endothelial cells. Confocal microscopic analysis of endothelial cells stained with monoclonal antibody TM-311, as well as biotin labeling of cell surface proteins on intact endothelial cells, revealed that tropomyosin exposure was enhanced on the surface of proliferating cells. These studies demonstrate that the antiangiogenic effects of HKa depend on high-affinity binding to endothelial cell tropomyosin.
Subject(s)
Angiogenesis Inhibitors/metabolism , Angiogenesis Inhibitors/pharmacology , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Kininogen, High-Molecular-Weight/metabolism , Kininogen, High-Molecular-Weight/pharmacology , Tropomyosin/metabolism , Allantois/blood supply , Allantois/drug effects , Animals , Antibodies, Monoclonal/pharmacology , Apoptosis/drug effects , Base Sequence , Cells, Cultured , Chick Embryo , Chorion/blood supply , Chorion/drug effects , DNA, Complementary/genetics , Endothelium, Vascular/cytology , Humans , Kininogen, High-Molecular-Weight/genetics , Neovascularization, Physiologic/drug effects , Protein Binding , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Tropomyosin/antagonists & inhibitorsABSTRACT
This study investigated the anti-angiogenic activities of Cnidium officinale Makino and Tabanus bovinus by using cultured glomerular capillary endothelial cells (GECs), chorioallantoic membrane (CAM) and rat cornea. Treatment of GECs with several concentrations (5-50 microg/ml) of C. officinale Makino and T. bovinus extracts for 24 h inhibited angiotensin II (10(-8) M)-induced increases of [3H]thymidine uptake and cell numbers in a concentration-dependent manner. The extent of inhibitory rate of [3H]thymidine incorporation by C. officinale Makino and T. bovinus at 50 microg/ml was a similar to that by 10(-5) M of retinoic acid. Herbal extracts also conspicuously inhibited the neovascularization. In contrast to the normal branching of vascular vessels, blood vessel patterns in CAMs treated with extracts (50 microg per egg) of C. officinale Makino and T. bovinus were ran parallel to each other without much branching. Moreover, oral administration of herbal extracts (20 mg/kg per day) for 4 weeks significantly inhibited the rat corneal neovascularization induced by suture, and the length of blood vessels in herbal medicine-treated rat cornea was conspicuously lower than that in control animals. A similar inhibitory effect to these was also observed in the rat cornea treated with thalidomide (200 mg/kg per day). These findings indicate that the anti-angiogenic properties of C. officinale Makino and T. bovinus may be one of the pharmacological mechanisms underlying the anti-tumor and anti-metastatic activities of herbal extracts tested in this study.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Cnidium/chemistry , Neovascularization, Physiologic/drug effects , Plant Extracts/pharmacology , Administration, Oral , Allantois/blood supply , Allantois/drug effects , Angiogenesis Inhibitors/administration & dosage , Animals , Antineoplastic Agents/pharmacology , Cell Division/drug effects , Cells, Cultured , Chick Embryo , Chorion/blood supply , Chorion/drug effects , Cornea/blood supply , Cornea/drug effects , Dose-Response Relationship, Drug , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Kidney Glomerulus/cytology , Kidney Glomerulus/drug effects , Male , Plant Extracts/administration & dosage , Rats , Rats, Sprague-DawleyABSTRACT
In this study the irritation phenomena at the chorioallantoic membrane of incubated hen's eggs as an in vitro model (HET-CAM assay) were investigated in comparison to the in vivo croton oil test by including hydrocortisone, indomethacin, phenylbutazone, acetylsalicylic acid, rutin, quercetin, apigenin, and p-coumaric acid as steroidal and non-steroidal test substances. For the first time the two methods were compared in a valid way with the perspective of a realistic reduction of animal experiments. It should be investigated whether an in vitro-in vivo correlation exists and, if there is any possibility, to replace the in vivo model by an in vitro test system. Both bioassays were able to demonstrate the anti-inflammatory potency of the constituents tested. The determination of the anti-inflammatory activity of all compounds in the two test systems showed individual trends of inhibitory effects. However, the in vitro HET-CAM test was much more sensitive in comparison to the in vivo croton oil test. The croton oil test gave dose-effect correlations in the anti-inflammatory substances investigated. The modified HET-CAM assay did not provide clear dose-effect ratios. The HET-CAM assay is an inexpensive test being easy to manage after a short practical training. Because of its sensitivity the HET-CAM assay could be considered a suitable tool for qualitative testing of the anti-inflammatory activity of substances if no appropriate dose-effect curves are required. From these results it can be concluded that the different courses of the dose-effect curves may be primarily due to different mechanisms of action.
Subject(s)
Animal Testing Alternatives , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Biological Assay/methods , Croton Oil , Steroids/analysis , Allantois/drug effects , Animals , Chick Embryo , Chorion/drug effects , Inflammation , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
PURPOSE: Because vascular occlusion has been observed as a consequence of photodynamic therapy (PDT), this method has been successfully used for the treatment of choroidal neovascularization (CNV) in age-related macular degeneration (AMD). However, most conventional photosensitizers, primarily developed for tumor PDT, lack selectivity for the targeting of neovascularization. An experimental model has been developed for drug screening of new photosensitizers for the treatment of CNV associated with AMD. It consists of intravenous (IV) injection of photosensitizers and fluorescent dyes into the chick's chorioallantoic membrane (CAM), followed by measurement of fluorescence pharmacokinetics, leakage from the vascular system, and photothrombic efficacy. METHODS: Fertilized chicken eggs were placed under a fluorescence microscope. After intravenous injection of different dyes, time-dependent fluorescence angiography was performed. The effect of PDT parameters was assessed by fluorescence angiography 24 hours after PDT. RESULTS: Although fluorescence of lipophilic benzoporphyrin derivative monoacid ring A (BPD-MA) remained intravascular during 2 hours, hydrophilic dyes tended to leak through the fenestrated neovascularization. By variation of PDT parameters, vascular damage could be directed toward closure of vessels with a diameter smaller than 10 microm, as measured 24 hours after PDT. High photosensitizer concentrations and high light doses resulted in blood flow stasis within 60 minutes, confirmed by fluorescence angiography. CONCLUSIONS: Fluorescence angiography and PDT after IV injection into the CAM showed strong similarities to results obtained in clinical tests of PDT in CNV associated with AMD. Thus, this model can provide valuable information about PDT mechanisms and can be used for drug-screening purposes in development of improved sensitizers for the PDT of CNV.