The role of PI3K/Akt/mTOR signaling in dose-dependent biphasic effects of glycine on vascular development.

Glycine, a non-essential amino acid, exerts concentration-dependent biphasic effects on angiogenesis. Low-doses of glycine promote angiogenesis, whereas high-doses cause anti-angiogenesis. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling participates in angiogenesis of both physiological development, and pathological events including tumor and inflammation. We assessed the role of PI3K/Akt/mTOR signaling in vascular development, and the interaction with glycine, using transgenic zebrafish Tg(fli1a:Myr-mCherry)ncv1 embryos expressing fluorescent proteins in vascular endothelial cells. Treatment with inhibitors of mTORC1 (rapamycin and everolimus), mTORC1/mTORC2 (KU0063794), PI3K (LY29400), and Akt (Akt inhibitor) decreased the development of intersegmental vessels (ISVs). These inhibitors cancelled the angiogenic effects of a low-dose of glycine, while acted synergistically with a high-dose of glycine in anti-angiogenesis. mTOR signaling regulates the gene expression of vascular endothelial growth factor (VEGF), a major angiogenic factor, and nitric oxide (NO) synthase (NOS), an enzyme for the synthesis of an angiogenic mediator NO. Expressions of VEGF and NOS were consistent with the vascular features induced by glycine and an mTOR inhibitor. Our results suggest that PI3K/Akt/mTOR signaling may interact with dose-dependent biphasic effects of exogenous glycine on in vivo angiogenesis. mTOR signaling is a key target for cancer therapy, thus, the combining mTOR inhibitors with glycine may be a potential approach for controlling angiogenesis.

Pro-angiogenic activity of Tongnao decoction on HUVECs in vitro and zebrafish in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Tongnao Decoction (TND) is a Chinese decoction approved and used in Jiangsu Province Hospital for the treatment of ischemic stroke. It shows conclusive efficiency in the improvement of neurologic impairment and activities of daily living of the patients. AIM OF THE STUDY: Recently, angiogenesis has been recognized as a potential therapeutic strategy for treating cerebral ischemia. This study was aimed to provide comprehensive evidence for the pro-angiogenic effect of TND and characterize the underlying mechanism. MATERIALS AND METHODS: We firstly established the chemical fingerprinting of TND. Then, the in vitro pro-angiogenic activities of TND were tested on human umbilical vein endothelial cells (HUVECs) through cell viability, wound healing and tube formation assays. The in vivo pro-angiogenic effects were evaluated on transgenic zebrafish embryos [Tg (fli-1: EGFP)] through the formation of intersegmental vessels (ISVs), subintestinal vessels (SIVs) and central arteries (CtAs). Lastly, the potential mechanisms of TND were analyzed by a blocking assay with eight pathways-specific kinase inhibitors. RESULTS: TND promoted the proliferation, migration and tube formation of HUVECs. TND also rescued the impairment of ISVs, SIVs and CtAs caused by VRI in a dose-dependent manner in zebrafish embryos. TND could activate vascular endothelial growth factor receptor-2 (VEGFR-2), phosphoinositide 3-kinase (PI3K) - protein kinase B (Akt) and Raf - mitogen-activated protein kinase1/2 (MEK1/2) - extracellular regulated kinase 1/2 (ERK1/2) signaling pathways. CONCLUSION: Our study firstly demonstrated the pro-angiogenic activities of TND. Our work provided evidences for the clinical usage of TND in restoring neurovascular function through promoting angiogenesis in the ischemic cerebral microvascular.

Crinamine Induces Apoptosis and Inhibits Proliferation, Migration, and Angiogenesis in Cervical Cancer SiHa Cells.

Crinumasiaticum is a perennial herb widely distributed in many warmer regions, including Thailand, and is well-known for its medicinal and ornamental values. Crinum alkaloids contain numerous compounds, such as crinamine. Even though its mechanism of action is still unknown, crinamine was previously shown to possess anticancer activity. In this study, we demonstrate that crinamine was more cytotoxic to cervical cancer cells than normal cells. It also inhibited anchorage-independent tumor spheroid growth more effectively than existing chemotherapeutic drugs carboplatin and 5-fluorouracil or the CDK9 inhibitor FIT-039. Additionally, unlike cisplatin, crinamine induced apoptosis without promoting DNA double-strand breaks. It suppressed cervical cancer cell migration by inhibiting the expression of positive regulators of epithelial-mesenchymal transition SNAI1 and VIM. Importantly, crinamine also exerted anti-angiogenic activities by inhibiting secretion of VEGF-A protein in cervical cancer cells and blood vessel development in zebrafish embryos. Gene expression analysis revealed that its mechanism of action might be attributed, in part, to downregulation of cancer-related genes, such as AKT1, BCL2L1, CCND1, CDK4, PLK1, and RHOA. Our findings provide a first insight into crinamine's anticancer activity, highlighting its potential use as an alternative bioactive compound for cervical cancer chemoprevention and therapy.

Antiangiogenic phenylpropanoid glycosides from Gynura cusimbua.

A new phenylpropanoid glycoside, named α-L-rhamnopyranosyl-(1↔2)-ß-D-[4″-(8E)-7-(3,4-dihydroxyphenyl)-8-propenoate, 1″-O-(7S)-7-(3,4-dihydroxyphenyl)-7-methoxy-ethyl]-glucopyranoside (1), together with nine known compounds (2-10) were isolated from the active fraction (n-Butanol fraction) of Gynura cusimbua for the first time. The known compounds (2-10) were identified as phenylpropanoid glycosides on the basis of extensive spectral data and references. The antiangiogenic activities of compounds (1-10) were evaluated by MTT assay on HUVECs and wild-type zebrafish in vivo model assay. As a result, compounds 1, 6, 7, 8 and 10 exhibited certain antiangiogenic activities.

Non-toxic and non teratogenic extract of Thuja orientalis L. inhibited angiogenesis in zebra fish and suppressed the growth of human lung cancer cell line.

Lung cancer is a malignant tumour with minimal survival rate and the current treatments are not showing complete remission of tumour and have many side effects. Thus a natural herbal medicine with good anti-cancer properties is highly demanded. Thuja orientalis L. is a traditionally used medicine to cure cough, bronchitis, excessive menstruation, asthma, skin infection and premature baldness. In addition, recent studies have revealed that it has anti-proliferative and anti-cancer activity. Angiogenesis is the main reason for the propagation and metastasis of cancers. We therefore intended to study the effects of the leaf extract of Thuja orientalis L. on angiogenesis as well as lung cancer cell growth. We have tested the anti-angiogenesis efficiency by alkaline phosphatase assay and also analysed the in vivo toxicity and teratogenic effects of various concentration of Thuja orientalis L. extract by establishing an in vivo zebra fish (Danio rerio), a promising model for cancer research which share genetic structure similarity to that of human beings. Also we demonstrated an anti-cancer effect of leaf extract from Thuja orientalis L. on human lung cancer cell line (A549) by MTT and trypan blue assay. The results revealed that the Thuja orientalis L. extract is efficient in repressing lung tumour cell growth significantly (p ≤ 0.01) in all treatments (2.4 mg/ml to 0.3 mg/ml) except 0.15 mg/ml compared to the control. The in vivo toxicity assay has proven that it is non-toxic at concentrations 0.6 mg/ml, 0.3 mg/ml and 0.15 mg/ml in zebrafish. The teratogenic assays revealed the therapeutic index (TI) as 0.808 with 0.7029 mg/ml as LC50 concentration at 24 h which is within the desirable value (below 1) for drug administration. Noticeable inhibition of angiogenesis also was observed in treatment with 2.4 mg/ml to 0.3 mg/ml. Overall we found that Thuja orientalis L. plant leaf extract exhibits better anti-cancer properties as we have validated by in vitro and in vivo analysis.

The extract of Polygoni Cuspidati Rhizoma et Radix suppresses the vascular endothelial growth factor-induced angiogenesis.

BACKGROUND: Polygoni Cuspidati Rhizoma et Radix (PCRR; the root and rhizome of Polygonum cuspidatum Sieb. et Zucc) is a traditional Chinese medicine for the treatment of inflammation, hyperlipemia, favus, jaundice and scald. HYPOTHESIS/PURPOSE: The extract of PCRR inhibits vascular endothelial growth factor (VEGF)-induced angiogenesis. The hypothesis is supported by analysis of PCRR extract and investigation of pharmacological role and signaling mechanism of PCRR extract in regulating angiogenic responses. STUDY DESIGN: The PCRR ethanolic extract was examined for its inhibitory effects on angiogenesis based on VEGF-treated human umbilical vein endothelial cells and in zebrafish model METHODS: The effects and signaling mechanism of a standardized ethanolic extract of PCRR were tested on cell proliferation, migration and tube formation in VEGF-treated human umbilical vein endothelial cells, and which was further validated in zebrafish embryo model. RESULTS: The treatment of PCRR extract in cultured endothelial cells inhibited VEGF-induced cell proliferation, cell migration and tube formation in a dose-dependent manner and also suppressed the formation of sub-intestinal vessels in zebrafish embryos. Moreover, the applied PCRR extract suppressed VEGF-induced phosphorylations of VEGF receptor 2 (VEGFR2) and JNK. Thus, the site of effect triggered by PCRR was proposed to be mediated by VEGFR2. To further support this notion, the phosphorylations of Erk, Akt and eNOS, induced by VEGF, were markedly reduced under the challenge of PCRR extract: the reductions were subsequently further decreased in the present of inhibitors of Erk, Akt and eNOS. In parallel, the formation of ROS induced by VEGF in cultured endothelial cells was markedly reduced in the present of PCRR extract. CONCLUSION: Collectively, our studies demonstrated the pharmacological role and signaling mechanism of PCRR in regulation of angiogenic responses, which supported further evaluation and development of PCRR as a potential therapeutic agent for the treatment and prevention of diseases related with angiogenesis.

Vertebrate embryos as tools for anti-angiogenic drug screening and function.

The development of new angiogenic inhibitors highlights a need for robust screening assays that adequately capture the complexity of vessel formation, and allow for the quantitative evaluation of the teratogenicity of new anti-angiogenic agents. This review discusses the use of screening assays in vertebrate embryos, specifically focusing upon chicken and zebrafish embryos, for the detection of anti-angiogenic agents.

Preclinical evaluations on the efficacy of a topical Chinese herbal formula for swelling control and pain relief.

ETHNOPHARMACOLOGICAL RELEVANCE: Patients suffering from musculoskeletal pain and swellings occupy many hospital beds and demand many rehabilitation facilities. Chinese Medicine is offering many alternatives to ameliorate pain and swelling. However, evidence-based scientific publications supporting their efficacy on pain relief are inadequate. The in vitro and in vivo efficacy of a topical use Chinese herbal bath formula (HB) on anti-inflammation and swelling control was studied. MATERIALS AND METHODS: The therapeutic mechanisms of HB were studied in vitro via anti-inflammatory and pro-angiogenic assays on RAW264.7 and HUVEC cells, respectively. Fibroblast proliferation was also studied with Hs27 cells. The in vivo angiogenic effect of HB was also studied using zebrafish model, while its efficacy of in vivo anti-Inflammation and swelling control were investigated using rat paw edema model. The affected paw was treated by immersing it in the HB or distilled water as control. The sensation of pain, change in paw thickness and inflammation marker in serum were analyzed. RESULTS: In the anti-inflammation assay, HB significantly inhibited nitrite release from RAW264.7 by 47.6% at 800 µg/ml. In the pro-angiogenic assays, it reduced wound area in HUVEC by 8.2% and increased tube formation of HUVEC by 11.5% at 300 µg/ml. HB also stimulated Hs27 proliferation up to 23.5% at 1200 µg/ml. It showed in vivo pro-angiogenic effect by increasing the mean sprout number in the embryos of zebrafish by 2.4 folds. The in vivo therapeutic effects of HB on edema was illustrated by the significant longer thermal withdrawal latency and thinner paw thickness compared with control. After 14 days of treatment, HB also reduced the IL-6 concentration in the serum of rat by 20.9% significantly. CONCLUSIONS: This study showed that HB is effective for swelling control and pain relief from edema due to its anti-inflammatory and pro-angiogenic properties.

Lignans from the stems and leaves of Brandisia hancei and their effects on VEGF-induced vascular permeability and migration of HRECs and DLAV formation in zebrafish.

In our continuing search for novel antiangiogenic agents, a new lignan glycoside, (7R,8R)-1-(4-O-ß-d-glucopyranosyl-3-methoxyphenyl)-2-{2-methoxy-4-[1-(E)-propene-3-ol]-phenoxyl}-propane-1,3-diol (1), along with three known lignans (2-4), were isolated from the 80% EtOH extract of Brandisia hancei stems and leaves. These isolates (1-4) were subjected to an in vitro bioassay to evaluate their effects on vascular endothelial growth factor (VEGF)-induced vascular permeability and migration of human retinal endothelial cells (HRECs). Of the compounds tested, compound 1 resulted in the greatest reduction in VEGF-induced vascular permeability by about 31.5% at 10 µM compared to the VEGF-treated control. In the migration assay, compounds 1 and 2 significantly decreased VEGF-induced HREC migration. Furthermore, zebrafish embryos treated with compounds 1 and 2 showed mild reductions of dorsal longitudinal anastomotic vessel (DLAV) formation.

A novel poly-naphthol compound ST104P suppresses angiogenesis by attenuating matrix metalloproteinase-2 expression in endothelial cells.

Angiogenesis, the process of neovascularization, plays an important role in physiological and pathological conditions. ST104P is a soluble polysulfated-cyclo-tetrachromotropylene compound with anti-viral and anti-thrombotic activities. However, the functions of ST104P in angiogenesis have never been explored. In this study, we investigated the effects of ST104P in angiogenesis in vitro and in vivo. Application of ST104P potently suppressed the microvessels sprouting in aortic rings ex vivo. Furthermore, ST104P treatment significantly disrupted the vessels' development in transgenic zebrafish in vivo. Above all, repeated administration of ST104P resulted in delayed tumor growth and prolonged the life span of mice bearing Lewis lung carcinoma. Mechanistic studies revealed that ST104P potently inhibited the migration, tube formation and wound closure of human umbilical endothelial cells (HUVECs). Moreover, ST104P treatment inhibited the secretion and expression of matrix metalloproteinase-2 (MMP-2) in a dose-dependent manner. Together, these results suggest that ST104P is a potent angiogenesis inhibitor and may hold potential for treatment of diseases due to excessive angiogenesis including cancer.

[Effect and mechanism of curcumol on angiogenesis activity of zebrafishes].

OBJECTIVE: To conduct a preliminary study on the effect of curcumol in promoting angiogenesis activity and its mechanism in zebrafishes, in order to provide basis for clinical prescription. METHOD: Zebrafishes biological model was established to, observe curcumol's effect on embryo blood vessel growth, blood vessel regeneration of adult fishes after tail-cutting and tissue regeneration of fish fries after tail-cutting. The relative fluorescence quantitative PCR method was adopted to determine the gene expression of vascular endothelial growth factor (VEGFA) and receptor VEGFR2 of fish fries after tail-cutting. RESULT: Curcumol contributed to angiogenesis of intersegmental blood vessels in zebrafishes embryos and speed up regeneration of blood vessels in adult fishes after tail-cutting. Furthermore, curcumol can increase the gene expression of VEGFA and VEGFR2 in fish fries. CONCLUSION: Curcumol can promote angiogenesis in zebrafishes, and enhance the gene expression of VEGFA and VEGFR2 in fish fries after tail-cutting and speed up the regeneration of their tails.

Angiogenic efficacy of simplified 2-herb formula (NF3) in zebrafish embryos in vivo and rat aortic ring in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Diabetic foot ulceration results in high risk of lower extremity amputation, and represents a significant health care expenditure worldwide. Radix Astragali (RA) and Radix Rehmanniae (RR) are widely used Chinese medicinal herbs in treating diabetes, and have shown positive effects in enhancing wound healing in diabetic foot ulcer animal model. MATERIALS AND METHODS: The angiogenic efficacy of NF3, a simplified 2-herb formula consisting of RA and RR in 2:1 ratio, was investigated. Median lethal concentration (LC50) and median effective concentration (EC50) were determined by treating zebrafish embryos with different concentrations of NF3 from 20 hpf to 72 hpf. The angiogenic activity of NF3 was examined in zebrafish embryos in vivo and by rat aortic ring assay in vitro. Cell cycle analysis of endothelial cells induced by NF3 was analyzed by flow cytometry using transgenic zebrafish Tg(fli1:EGFP). Real-time PCR was used to analyze mRNA expression profiles of selected genes involved in VEGF, FGF and MAPK pathways. RESULTS: NF3 enhanced blood vessel formation as indicated by extra growth of intersegmental vessels in zebrafish embryos, and increased microvessels formation in rat aortic ring. NF3 also enhanced endothelial cells proliferation as shown by increased percentage of cells accumulating in S phase and G2/M phase of the cell cycle. NF3 exposure significantly induced up-regulation of VEGF-A, Flk-1, fgf1 and bRaf expression in zebrafish embryos. CONCLUSIONS: Our results demonstrated that NF3 was effective in promoting angiogenesis in zebrafish embryos and by rat aortic ring assay, which provided scientific basis to support the use of NF3 as potential therapeutics in treating diabetic foot ulceration.

Transcriptional profiling of angiogenesis activities of calycosin in zebrafish.

Angiogenesis plays an important role in a wide range of physiological processes and many diseases are associated with the dysregulation of angiogenesis. The commonly used Chinese herbal medicine Radix Astragali (known as Huang qi in Chinese) is a potential candidate for treating this type of disease. Calycosin, a major isoflavonoid in Radix Astragali, was identified in our earlier study and shown to induce angiogenesis in human umbilical vein endothelial cells (HUVEC) in vitro and in zebrafish embryos in vivo. Using zebrafish as a testing model, we investigated the angiogenic effect of calycosin on the subintestinal vessels (SIVs) in zebrafish embryos. Our findings using transcriptional profiling by deep sequencing, and confirmed by quantitative real-time PCR (qPCR), demonstrate that calycosin modulated vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and ErbB signaling pathways. The inhibitory effects of calycosin-induced phenotypic responses by several pathway-specific inhibitors (VRI, SU5402, MEK1/2 Inhibitor, Wortmannin and LY294002) further identified the potential involvement of VEGF(R) and FGF(R) signaling pathways in the angiogenic activities of calycosin. We present a comprehensive framework of study using fluorescence microscopy, transcriptomics and qPCR to demonstrate the proangiogenic effects of calycosin in vivo. The data have elucidated the connection between morphological observations and genomic evidence, indicating the potential roles of several key signaling pathways in angiogenesis.

Bioassay-guided isolation of norviburtinal from the root of Rehmannia glutinosa, exhibited angiogenesis effect in zebrafish embryo model.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Rehmannia glutinosa (RR) is commonly used as a wound-healing agent in various traditional Chinese herbal formulae; while angiogenesis is one of the crucial aspects in wound-healing. AIM OF THE STUDY: The objective of the present study was to investigate the angiogenesis effects of RR aqueous crude extract and its active component(s) using zebrafish model. MATERIALS AND METHODS: The in vivo angiogenesis effect was studied using transgenic TG(fli1:EGFP)(y1)/+(AB) zebrafish embryos by observing the capillary sprouts formation in sub-intestinal vessel (SIV) of zebrafish embryos after 72 h post-fertilization under fluorescence microscopy. RESULTS: Our results indicated that RR aqueous crude extract (250 µg/ml) exhibited significant angiogenesis effect, with an increase in capillary sprouts formation in SIV. Following sequential solvent partition of the RR aqueous crude extract with dichloromethane, ethyl acetate and n-butanol successively, the dichloromethane fraction (DCM) was found to have the most sprouts formation in the SIV region. Subjected to column chromatography, DCM fraction was further fractionated into six sub-fractions and among these tested, the sub-fraction C2 exhibited the most potent angiogenesis effect. The major component, C2A, was isolated and identified as norviburtinal using nuclear magnetic resonance (NMR) and mass spectrometry (MS). The compound norviburtinal (at 50 µg/ml) was shown to possess significant angiogenesis effect in zebrafish model (p < 0.001). CONCLUSIONS: Norviburtinal was, for the first time, found in the extract of RR and possessed novel angiogenesis effect. Bioassay-guided fractionation suggested that norviburtinal was not the only active component responsible for the angiogenesis effect of RR.

Polyphyllin D, a steroidal saponin from Paris polyphylla, inhibits endothelial cell functions in vitro and angiogenesis in zebrafish embryos in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Angiogenesis, the process of blood vessel formation, is critical to tumour growth. The importance of angiogenesis in tumour development has lead to the development of anti-angiogenic strategies to inhibit tumour growth. In this study, polyphyllin D (PD), an active component in Chinese herb, Paris polyphylla, was evaluated for its potential anti-angiogenic effects. MATERIALS AND METHODS: The inhibitory effects of PD on three important processes involved in angiogenesis, i.e. proliferation, migration and differentiation were examined using human microvascular endothelial cell line HMEC-1 by MTT assay, scratch assay and tube formation assay, respectively. Using zebrafish embryos as an animal model of angiogenesis, the anti-angiogenic effect of PD was further verified in vivo. RESULTS: PD suppressed the growth of HMEC-1 cells at 0.1-0.4 µM without toxic effects. At 0.3 µM and 0.4 µM, PD significantly inhibited endothelial cell migration and capillary tube formation. About 70% of the zebrafish embryos showed defects in intersegmental vessel formation upon treatment with PD at concentrations of 0.156 µM and 0.313 µM. CONCLUSION: The anti-angiogenic effects of PD have been explored in the study which implied a potential therapeutic development of PD in cancer treatment.

Zebrafish bioassay-guided natural product discovery: isolation of angiogenesis inhibitors from East African medicinal plants.

Natural products represent a significant reservoir of unexplored chemical diversity for early-stage drug discovery. The identification of lead compounds of natural origin would benefit from therapeutically relevant bioassays capable of facilitating the isolation of bioactive molecules from multi-constituent extracts. Towards this end, we developed an in vivo bioassay-guided isolation approach for natural product discovery that combines bioactivity screening in zebrafish embryos with rapid fractionation by analytical thin-layer chromatography (TLC) and initial structural elucidation by high-resolution electrospray mass spectrometry (HRESIMS). Bioactivity screening of East African medicinal plant extracts using fli-1:EGFP transgenic zebrafish embryos identified Oxygonum sinuatum and Plectranthus barbatus as inhibiting vascular development. Zebrafish bioassay-guided fractionation identified the active components of these plants as emodin, an inhibitor of the protein kinase CK2, and coleon A lactone, a rare abietane diterpenoid with no previously described bioactivity. Both emodin and coleon A lactone inhibited mammalian endothelial cell proliferation, migration, and tube formation in vitro, as well as angiogenesis in the chick chorioallantoic membrane (CAM) assay. These results suggest that the combination of zebrafish bioassays with analytical chromatography methods is an effective strategy for the rapid identification of bioactive natural products.

Antiangiogenic activity of Tripterygium wilfordii and its terpenoids.

ETHNOPHARMACOLOGICAL RELEVANCE: Tripterygium wilfordii Hook. f. (Celastraceae) has been traditionally used as folk medicine for centuries in China for the treatment of immune-inflammatory diseases. AIM OF THE STUDY: This study aimed to assess the antiangiogenic activities which support the therapeutic use of Tripterygium wilfordii and its terpenoids for angiogenesis disease such as cancer. MATERIALS AND METHODS: The ethanol extract of Tripterygium wilfordii and subsequent fractions were evaluated on an in vivo antiangiogenic zebrafish embryo model. RESULTS: Three antiangiogenic terpenoids were isolated by bioassay-guided purification, namely, celastrol (4), cangoronine (5) and triptolide (7). Among them, triptolide manifested the most potent antiangiogenic activity against vessel formation by nearly 50% at 1.2 microM. Semi-quantitative RT-PCR analysis revealed that triptolide dose- and time-dependently reduced the mRNA expression of angiopoietin (angpt)2 and tie2 in zebrafish, indicating the involvement of angpt2/tie2 signaling pathway in the antiangiogenic action of triptolide. CONCLUSIONS: The discovery of an alternative pathway further confirms the value of ethnopharmacological investigations into traditional botanicals for leads for potential drug development.

Dissection of angiogenic signaling in zebrafish using a chemical genetic approach.

Striking homology between signaling molecules in zebrafish and humans suggests that compounds known to inhibit human kinases may enable a chemical genetic approach to dissect signaling pathways in the zebrafish embryo. We tested this hypothesis using a vascular endothelial growth factor receptor inhibitor, PTK787/ZK222584. Zebrafish embryos treated with this compound lacked all major blood vessels. Overexpression of AKT/PKB, a putative effector of vascular endothelial growth factor signaling, allowed blood vessels to form in the presence of drug. Endothelial cell apoptosis induced by the drug is prevented by increasing AKT/PKB activity, thus establishing the physiological relevance of AKT/PKB in the angiogenic process. This approach allowed us to examine the effects of blood flow and the role of endothelial signals in organogenesis.

Effect of supplemental oxygen on blood plasma organic acids within embryos from selected lines of turkeys.

The hypothesis was tested that increased oxygen tensions during the plateau stage of oxygen consumption (25 and 26 d of incubation) would cause different metabolic responses from embryos selected for increased egg production or growth. Embryos were exposed to 171 or 152 mm Hg partial pressure of oxygen from 25 to 28 d of incubation, a time when the oxygen conductance properties of the eggshell are exceeded by the embryonic tissue demands for oxygen. Carbohydrate and lipid metabolism were observed by measuring plasma organic acids in embryos from selected lines and randombred controls. (E was selected for increased egg production; RBC1 is the randombred line from which it was selected. F was selected for increased BW; RBC2 is the randombred line from which it was selected.) During the plateau stage in oxygen consumption, RBC2 embryos responded to added oxygen by utilizing fat rather than carbohydrate, whereas F embryos responded by using less fat as well as less carbohydrate from the liver and kidney. The response of F embryos to added oxygen is the opposite that might be expected for aerobic metabolism. The reason that selection for growth has resulted in such a metabolism is unknown. The E embryos displayed depressed lactate and beta-hydroxybutyrate levels, but plasma urates were elevated compared with RBC1, suggesting that the selection for egg production has also resulted in a unique metabolism. The embryonic processes described in the current study suggest that selected embryos are unable to respond to elevated partial pressure of oxygen by adjusting energy metabolism, which may result in increased embryonic mortality during this stage.