The relationship between plastic ingestion and trace element concentrations in Arctic seabirds.

Seabirds ingest contaminants linked to their prey's tissues, but also adsorbed to ingested plastic debris. To explore relationships between ingested plastics and trace elements concentrations, we analyzed 25 essential non-essential trace elements in liver tissue in relation to plastic content in the gastrointestinal tract in adults of four species of Arctic seabirds with different propensity to ingest plastic. Linear Discriminant Analysis (LDA) provided a clear separation between species based on element concentrations, but not among individuals with and without plastics. Molybdenum, copper, vanadium, and zinc were strong drivers of the LDA, separating northern fulmars (Fulmarus glacialis) from other species (60.4 % of explained between-group variance). Selenium, vanadium, zinc, and mercury were drivers separating black-legged kittiwakes (Rissa tridactyla) from the other species (19.3 % of explained between-group variance). This study suggests that ingestion of plastic particles has little influence on the burden of essential and non-essential trace elements in Arctic seabird species.

Female and male Leach's Storm Petrels (Hydrobates leucorhous) pursue different foraging strategies during the incubation period.

Reproduction in procellariiform birds is characterized by a single egg clutch, slow development, a long breeding season and obligate biparental care. Female Leach's Storm Petrels Hydrobates leucorhous, nearly monomorphic members of this order, produce eggs that are between 20 and 25% of adult body weight. We tested whether female foraging behaviour differs from male foraging behaviour during the ~ 44-day incubation period across seven breeding colonies in the Northwest Atlantic. Over six breeding seasons, we used a combination of Global Positioning System and Global Location Sensor devices to measure characteristics of individual foraging trips during the incubation period. Females travelled significantly greater distances and went farther from the breeding colony than did males on individual foraging trips. For both sexes, the longer the foraging trip, the greater the distance. Independent of trip duration, females travelled farther, and spent a greater proportion of their foraging trips prospecting widely as defined by behavioural categories derived from a Hidden Markov Model. For both sexes, trip duration decreased with date. Sex differences in these foraging metrics were apparently not a consequence of morphological differences or spatial segregation. Our data are consistent with the idea that female foraging strategies differed from male foraging strategies during incubation in ways that would be expected if females were still compensating for egg formation.

Factors influencing mercury levels in Leach's storm-petrels at northwest Atlantic colonies.

Mercury (Hg) is a globally distributed heavy metal, with negative effects on wildlife. Its most toxic form, methylmercury (MeHg), predominates in aquatic systems. Levels of MeHg in marine predators can vary widely among individuals and populations. Leach's storm-petrels (Hydrobates leucorhous) have elevated levels of Hg but the role of Hg in storm-petrel population declines is unknown. In this study, we used egg and blood samples to study variation in Hg exposure among several northwest Atlantic colonies during breeding seasons, thereby evaluating relative toxicity risk within and among colonies. Total mercury (THg) concentrations were higher with increasing colony latitude, and were more pronounced in blood than in eggs. THg concentrations in blood were mostly associated with low toxicity risk in birds from the southern colonies and moderate risks in birds from the northern colonies; however, those values did not affect hatching or fledging success. THg concentrations in both eggs and blood were positively correlated with δ34S, emphasizing the role of sulfate-reducing bacteria in methylation of THg acquired through marine food webs, which is consistent with enriched δ34S profiles. By associating tracking data from foraging trips with THg from blood, we determined that blood THg levels were higher when storm-petrel's intensive search locations were over deeper waters. We conclude that spatial variation in THg concentrations in Leach's storm-petrels is attributable to differences in ocean depth at foraging locations, both at individual and colony levels. Differences in diet among colonies observed previously are the most likely cause for observed blood THg differences. As one of the few pelagic seabird species breeding in Atlantic Canada, with limited overlap in core foraging areas among colonies, Leach's storm-petrels can be used as biomonitors for less sampled offshore pelagic regions. The global trend in Hg emissions combined with legacy levels warrant continued monitoring for toxicity effects in seabirds.

Mercury levels in North Atlantic seabirds: A synthesis.

Mercury (Hg) is globally-distributed, with severe toxic effects on wildlife. Methylmercury biomagnifies within food webs, so long-lived, top predators such as seabirds are prone to high mercury concentrations. We synthesized historical and contemporary data on mercury concentrations in seabirds from the North Atlantic. We collected 614 values determined from 39 species and 115 locations, ranging from 1895 to 1940 and from 1970 to 2020. Highest blood-equivalent Hg values were in Phalacrocoracidae. For the same species/tissue/collection site, blood-equivalent values were lower during pre-1940 than post-1970 period. In almost 5 % of post-1970 values, mean blood-equivalent Hg concentrations were above those considered to pose severe risks of adverse effects, and 21 % were above the high-risk effect. We found an imbalance in sample effort and did not find Hg values for many species. We argue that stronger, trans-Atlantic Hg monitoring schemes are required to coordinate research and better compare trends across a wide scale.

Tapping into non-English-language science for the conservation of global biodiversity.

The widely held assumption that any important scientific information would be available in English underlies the underuse of non-English-language science across disciplines. However, non-English-language science is expected to bring unique and valuable scientific information, especially in disciplines where the evidence is patchy, and for emergent issues where synthesising available evidence is an urgent challenge. Yet such contribution of non-English-language science to scientific communities and the application of science is rarely quantified. Here, we show that non-English-language studies provide crucial evidence for informing global biodiversity conservation. By screening 419,679 peer-reviewed papers in 16 languages, we identified 1,234 non-English-language studies providing evidence on the effectiveness of biodiversity conservation interventions, compared to 4,412 English-language studies identified with the same criteria. Relevant non-English-language studies are being published at an increasing rate in 6 out of the 12 languages where there were a sufficient number of relevant studies. Incorporating non-English-language studies can expand the geographical coverage (i.e., the number of 2° × 2° grid cells with relevant studies) of English-language evidence by 12% to 25%, especially in biodiverse regions, and taxonomic coverage (i.e., the number of species covered by the relevant studies) by 5% to 32%, although they do tend to be based on less robust study designs. Our results show that synthesising non-English-language studies is key to overcoming the widespread lack of local, context-dependent evidence and facilitating evidence-based conservation globally. We urge wider disciplines to rigorously reassess the untapped potential of non-English-language science in informing decisions to address other global challenges. Please see the Supporting information files for Alternative Language Abstracts.

Year-round spatiotemporal distribution pattern of a threatened sea duck species breeding on Kolguev Island, south-eastern Barents Sea.

BACKGROUND: The long-tailed duck (Clangula hyemalis) was categorized as ´Vulnerable` by the IUCN after a study revealed a rapid wintering population decline of 65% between 1992-1993 and 2007-2009 in the Baltic Sea. As knowledge about the European long-tailed duck's life cycle and movement ecology is limited, we investigate its year-round spatiotemporal distribution patterns. Specifically, we aimed to identify the wintering grounds, timing of migration and staging of this population via light-level geolocation. RESULTS: Of the 48 female long-tailed ducks tagged on Kolguev Island (western Russian Arctic), 19 were recaptured to obtain data. After breeding and moulting at freshwater lakes, ducks went out to sea around Kolguev Island and to marine waters ranging from the White Sea to Novaya Zemlya Archipelago for 33 ± 10 days. After a rapid autumn migration, 18 of 19 birds spent their winter in the Baltic Sea and one bird in the White Sea, where they stayed for 212 ± 3 days. There, they used areas known to host long-tailed ducks, but areas differed among individuals. After a rapid spring migration in mid-May, the birds spent 23 ± 3 days at sea in coastal areas between the White Sea and Kolguev Island, before returning to their freshwater breeding habitats in June. CONCLUSIONS: The Baltic Sea represents the most important wintering area for female long-tailed ducks from Kolguev Island. Important spring and autumn staging areas include the Barents Sea and the White Sea. Climate change will render these habitats more exposed to human impacts in the form of fisheries, marine traffic and oil exploitation in near future. Threats that now operate in the wintering areas may thus spread to the higher latitude staging areas and further increase the pressure on long-tailed ducks.

Foraging areas, offshore habitat use, and colony overlap by incubating Leach's storm-petrels Oceanodroma leucorhoa in the Northwest Atlantic.

Despite their importance in marine food webs, much has yet to be learned about the spatial ecology of small seabirds. This includes the Leach's storm-petrel Oceanodroma leucorhoa, a species that is declining throughout its Northwest Atlantic breeding range. In 2013 and 2014, we used global location sensors to track foraging movements of incubating storm-petrels from 7 eastern Canadian breeding colonies. We determined and compared the foraging trip and at-sea habitat characteristics, analysed spatial overlap among colonies, and determined whether colony foraging ranges intersected with offshore oil and gas operations. Individuals tracked during the incubation period made 4.0 ± 1.4 day foraging trips, travelling to highly pelagic waters over and beyond continental slopes which ranged, on average, 400 to 830 km from colonies. Cumulative travel distances ranged from ~900 to 2,100 km among colonies. While colony size did not influence foraging trip characteristics or the size of areas used at sea, foraging distances tended to be shorter for individuals breeding at the southern end of the range. Core areas did not overlap considerably among colonies, and individuals from all sites except Kent Island in the Bay of Fundy foraged over waters with median depths > 1,950 m and average chlorophyll a concentrations ≤ 0.6 mg/m3. Sea surface temperatures within colony core areas varied considerably (11-23°C), coincident with the birds' use of cold waters of the Labrador Current or warmer waters of the Gulf Stream Current. Offshore oil and gas operations intersected with the foraging ranges of 5 of 7 colonies. Three of these, including Baccalieu Island, Newfoundland, which supports the species' largest population, have experienced substantial declines in the last few decades. Future work should prioritize modelling efforts to incorporate information on relative predation risk at colonies, spatially explicit risks at-sea on the breeding and wintering grounds, effects of climate and marine ecosystem change, as well as lethal and sub-lethal effects of environmental contaminants, to better understand drivers of Leach's storm-petrel populations trends in Atlantic Canada.

Relationships between blood mercury levels, reproduction, and return rate in a small seabird.

Mercury (Hg) is a ubiquitous heavy metal that occurs naturally in the environment, but its levels have been supplemented for decades by a variety of human activities. Mercury can have serious deleterious effects on a variety of organisms, with top predators being particularly susceptible because methylmercury bioaccumulates and biomagnifies in food webs. Among birds, seabirds can have especially high levels of Hg contamination and Leach's storm-petrels (Oceanodroma leucorhoa), in particular, have amongst the highest known levels. Several populations of Leach's storm-petrels have declined recently in the Northwest Atlantic. The causes of these declines remain uncertain, but the toxic effects of Hg could be a potential factor in this decline. Here, we tested for relationships between adult blood total Hg (THg) concentration and several offspring development parameters, and adult return rate of Leach's storm-petrels breeding on Bon Portage Island (43° 28' N, 65° 44' W), Nova Scotia, Canada, between 2011 and 2015 (blood samples n = 20, 36, 6, 15, and 13 for each year, respectively). Overall, THg levels were elevated (0.78 ± 0.43 µg/g wet wt.) compared to other species of seabirds in this region, and varied significantly among years. However, we found no associations between THg levels and reproductive parameters or adult return rate. Our results indicate that levels of mercury observed in Leach's storm-petrel blood, although elevated, appear not to adversely affect their offspring development or adult return rate on Bon Portage Island.

American dippers indicate contaminant biotransport by Pacific salmon.

Migrating salmon can increase productivity in Pacific Northwestern streams and lakes through the deposition of nutrients from their decomposing carcasses after spawning. Several studies also report simultaneous biotransport of persistent organic pollutants that have contaminated lake food webs, although no similar effect has been shown conclusively in rivers. We tested the prediction that salmon enhance contaminants in river food webs using the American dipper (Cinclus mexicanus), an aquatic songbird and a recognized indicator of stream quality. Over 3 years, we analyzed 29 dipper eggs and aquatic invertebrate samples from 14 different rivers in 10 catchments in southern British Columbia, Canada to assess whether variations in autumn spawning density of Pacific salmon were reflected in dipper egg contamination or stable carbon and nitrogen isotopes. δ(13)C isotope signatures, but not δ(15)N, in aquatic invertebrates and dipper eggs increased among catchments in proportion to the average density of spawning salmon. Concentrations of brominated flame retardants (PBDEs), dichlorodiphenyltrichloroethane metabolites (DDTs), and chlordane compounds were related in part to the δ(13)C measure of salmon density, but mercury, chlorobenzenes, and polychlorinated biphenyls (PCBs) were explained better by dipper trophic level. We conclude that spawning Pacific salmon result in the increased availability of salmon fry as dipper prey and salmon are a significant source of PBDEs, DDTs, and chlordanes to river ecosystems. However, contrary to lake studies, postspawn concentrations of legacy PCBs in river birds, even in salmon-rich rivers, were not significantly higher than would be expected from atmospheric deposition alone. We recommend using δ(13)C isotopes to trace salmon-derived lipids which may persist over winter particularly in rivers, and are potentially a better reflection of lipophilic contaminant transfer.

Characterisation of a DNAPL source zone in a porous aquifer using the Partitioning Interwell Tracer Test and an inverse modelling approach.

In this paper, we discuss the results of a Partitioning Interwell Tracer Test (PITT) performed in a large scale experiment with a well-defined TCE spill, and present a novel combined analytical-numerical inverse modelling approach using measured concentration profiles within a TCE plume to predict the distribution of the DNAPL in a virtual vertical plane of the source. The proposed inverse modelling approach assumes local thermodynamic equilibrium of the distribution of TCE between the NAPL phase and the aqueous phase and no decay or sorption of the dissolved TCE concentrations downstream of the spill area. The analytical part of the inverse modelling approach contains two steps. As a first step, the location of the contaminant in a virtual vertical plane of a porous medium is fixed by using measured concentration profiles and considering the dissolution of the organic phase under equilibrium conditions. In the second step, the volume of contaminant entrapped in the source cells is estimated. A multiphase advective-dispersive transport model is used in the final step to adjust the volumes quantified in the second step. The predictions are highly dependent on the quantity and quality of the data in space and time. From the PITT-breakthrough curves measured at the pumping well, a mean TCE saturation in the sweep zone of 0.0004 was derived, which is very low compared to that determined at the local scale. In a second analysis, tracer breakthrough curves available at measuring points placed closely downstream and upstream of the presumed source zone, were used to explain why the globally obtained DNAPL saturation was very low compared to the "real", locally evaluated TCE saturations in the source zone. This was principally caused by the overall travel time compared to the short travel time of the tracers in the source zone. Another reason is that due to bypassing, only part of the volume of tracer injected had been in contact and had eventually interacted with the DNAPL. Furthermore, the quantified TCE volume was nearly 30% higher than the spilled volume; this agrees with the conclusions from other studies emphasizing that calculated volumes can overestimate the measured volumes, particularly in the case of an inhomogeneous distribution of the DNAPL within the soil. A good agreement of the measured and inversed concentration profiles was obtained, highlighting that it is possible to determine the length-averaged distribution of a residual pollution source from dissolved concentration profiles measured downstream of the source zone. The numerically obtained non-uniform distribution of DNAPL entrapped in the vertical plane of the source zone was experimentally confirmed by the TCE saturation values derived from PITT-breakthrough curves at measuring points located 0.75 m downstream of the source zone. However, the sensitivity study showed that the inverse modelling approach provided a rather non-unique solution. More data available may reduce the number of possible representations of the estimated source zone.

Jagged1-mediated Notch activation induces epithelial-to-mesenchymal transition through Slug-induced repression of E-cadherin.

Aberrant expression of Jagged1 and Notch1 are associated with poor outcome in breast cancer. However, the reason that Jagged1 and/or Notch overexpression portends a poor prognosis is unknown. We identify Slug, a transcriptional repressor, as a novel Notch target and show that elevated levels of Slug correlate with increased expression of Jagged1 in various human cancers. Slug was essential for Notch-mediated repression of E-cadherin, which resulted in beta-catenin activation and resistance to anoikis. Inhibition of ligand-induced Notch signaling in xenografted Slug-positive/E-cadherin-negative breast tumors promoted apoptosis and inhibited tumor growth and metastasis. This response was associated with down-regulated Slug expression, reexpression of E-cadherin, and suppression of active beta-catenin. Our findings suggest that ligand-induced Notch activation, through the induction of Slug, promotes tumor growth and metastasis characterized by epithelial-to-mesenchymal transition and inhibition of anoikis.

Identification of sokotrasterol sulfate as a novel proangiogenic steroid.

The potential to promote neovascularization in ischemic tissues using exogenous agents has become an exciting area of therapeutics. In an attempt to identify novel small molecules with angiogenesis promoting activity, we screened a library of natural products and identified a sulfated steroid, sokotrasterol sulfate, that induces angiogenesis in vitro and in vivo. We show that sokotrasterol sulfate promotes endothelial sprouting in vitro, new blood vessel formation on the chick chorioallantoic membrane, and accelerates angiogenesis and reperfusion in a mouse hindlimb ischemia model. We demonstrate that sulfation of the steroid is critical for promoting angiogenesis, as the desulfated steroid exhibited no endothelial sprouting activity. We thus developed a chemically synthesized sokotrasterol sulfate analog, 2beta,3alpha,6alpha-cholestanetrisulfate, that demonstrated equivalent activity in the hindlimb ischemia model and resulted in the generation of stable vessels that persisted following cessation of therapy. The function of sokotrasterol sulfate was dependent on cyclooxygenase-2 activity and vascular endothelial growth factor induction, as inhibition of either cyclooxygenase-2 or vascular endothelial growth factor blocked angiogenesis. Surface expression of alpha(v)beta(3) integrin was also necessary for function, as neutralization of alpha(v)beta(3) integrin, but not beta(1) integrin, binding abrogated endothelial sprouting and antiapoptotic activity in response to sokotrasterol sulfate. Our findings indicate that sokotrasterol sulfate and its analogs can promote angiogenesis in vitro and in vivo and could potentially be used for promoting neovascularization to relieve the sequelae of vasoocclusive diseases.

Minimal contribution of marrow-derived endothelial precursors to tumor vasculature.

During embryogenesis, vascular and hemopoietic cells originate from a common precursor, the hemangioblast. Recent evidence suggests the existence of endothelial precursors in adult bone marrow cells, but it is unclear whether those precursors have a role in tumor neovascularization. In this report, we demonstrate that murine bone marrow contains endothelial progenitors, which arise from a cell with self-renewing capacity, and can integrate into tumor microvasculature, albeit at a very low frequency. A transgenic double-reporter strategy allowed us to demonstrate definitively that tumor bone marrow-derived endothelial cells arise by transdifferentiation of marrow progenitors rather than by cell fusion. Single cell transplants showed that a common precursor contributes to both the hemopoietic and endothelial lineages, thus demonstrating the presence of an adult hemangioblast. Furthermore, we demonstrate that increased vascular endothelial growth factor (VEGF)-A secretion by tumor cells, as well as activation of VEGF receptor-2 in bone marrow cells does not alter the mobilization and incorporation of marrow-derived endothelial progenitors into tumor vasculature. Finally, in human umbilical cord blood cells, we show that endothelial precursors make up only approximately 1 in 10(7) mononuclear cells but are highly enriched in the CD133+ cell population. By ruling out cell fusion, we clearly demonstrate the existence of an adult hemangioblast, but the differentiation of marrow stem cells toward the endothelial lineage is an extremely rare event. Furthermore, we show that VEGF-A stimulation of hemopoietic cells does not significantly alter this process.

Activation of vascular endothelial growth factor receptor-2 in bone marrow leads to accumulation of myeloid cells: role of granulocyte-macrophage colony-stimulating factor.

Vascular endothelial growth factor (VEGF) is a secreted cytokine that plays a major role in the formation and maintenance of the hemopoietic and vascular compartments. VEGF and its receptors, VEGFR-1 and VEGFR-2, have been found to be expressed on subsets of normal and malignant hemopoietic cells, but the role of the individual receptors in hemopoiesis requires further study. Using a VEGFR-2 fusion protein that can be dimerized with a synthetic drug, we were able to specifically examine the effects of VEGFR-2 signaling in hemopoietic cells in vivo. Mice transplanted with bone marrow transduced with this inducible VEGFR-2 fusion protein demonstrated expansion of myeloid cells (Gr-1+, CD11b+). Levels of myeloid progenitors were also increased following VEGFR-2 activation, through autocrine and paracrine mechanisms, as measured by clonogenic progenitor assays. VEGFR-2 activation induced expression of GM-CSF and increased serum levels in vivo. Abrogation of GM-CSF activity, either with neutralizing Abs or by using GM-CSF-null hemopoietic cells, inhibited VEGFR-2-mediated myeloid progenitor activity. Our findings indicate that VEGF signaling through VEGFR-2 promotes myelopoiesis through GM-CSF-dependent and -independent mechanisms.

Quantitative analysis of the low molecular weight serum proteome using 18O stable isotope labeling in a lung tumor xenograft mouse model.

With advancements in the analytical technologies and methodologies in proteomics, there is great interest in biomarker discovery in biofluids such as serum and plasma. Current hypotheses suggest that the low molecular weight (LMW) serum proteome possesses an archive of clipped and cleaved protein fragments that may provide insight into disease development. Though these biofluids represent attractive samples from which new and more accurate disease biomarkers may be found, the intrinsic person-to-person variability in these samples complicates their discovery. Mice are one of the most extensively used animal models for studying human disease because they represent a highly controllable experimental model system. In this study, the LMW serum proteome was compared between xenografted tumor-bearing mice and control mice by differential labeling utilizing trypsin-mediated incorporation of the stable isotope of oxygen, 18O. The digestates were combined, fractionated by strong cation exchange chromatography, and analyzed by nanoflow reversed-phase liquid chromatography coupled online with tandem mass spectrometry, resulting in the identification of 6003 proteins identified by at least a single, fully tryptic peptide. Almost 1650 proteins were identified and quantitated by two or more fully tryptic peptides. The methodology adopted in this work provides the means for future quantitative measurements in comparative animal models of disease and in human disease cohorts.

Quantitative proteomic analysis of sokotrasterol sulfate-stimulated primary human endothelial cells.

The endothelium forms a continuous monolayer at the interface between blood and tissue and contributes significantly to the sensing and transducing of signals between blood and tissue. New blood vessel formation, or angiogenesis, is initiated by the activation of endothelial cells and is an important process required for various pathological and physiological situations. This study used cleavable isotope-coded affinity tag reagents combined with mass spectrometry to investigate the molecular basis of a recently discovered angiogenesis-promoting steroid, sokotrasterol sulfate. Changes in the relative abundances of over 1000 proteins within human endothelial cells treated with sokotrasterol sulfate and vehicle-treated cells were identified and quantitated using this technique. A method that examines the entire ensemble of quantitative measurements was developed to identify proteins that showed a statistically significant change in relative abundance resulting from treatment with sokotrasterol sulfate. A total of 93 proteins was significantly up-regulated, and 37 were down-regulated in response to sokotrasterol sulfate stimulation of endothelial cells. Among the up-regulated proteins, several were identified that are novel to endothelial cells and are likely involved in cell communication and morphogenesis. These findings are consistent with a role for sokotrasterol sulfate in endothelial sprouting.

Notch4-induced inhibition of endothelial sprouting requires the ankyrin repeats and involves signaling through RBP-Jkappa.

Notch proteins comprise a family of transmembrane receptors. Ligand activation of Notch releases the intracellular domain of the receptor that translocates to the nucleus and regulates transcription through the DNA-binding protein RBP-Jkappa. Previously, it has been shown that the Notch4 intracellular region (N4IC) can inhibit endothelial sprouting and angiogenesis. Here, N4IC deletion mutants were assessed for their ability to inhibit human microvascular endothelial cell (HMEC) sprouting with the use of a quantitative endothelial sprouting assay. Deletion of the ankyrin repeats, but not the RAM (RBP-Jkappa associated module) domain or C-terminal region (CT), abrogated the inhibition of fibroblast growth factor 2 (FGF-2)- and vascular endothelial growth factor (VEGF)-induced sprouting by Notch4, whereas the ankyrin repeats alone partially blocked sprouting. The ankyrin repeats were also the only domain required for up-regulation of RBP-Jkappa-dependent gene expression. Interestingly, enforced expression of the ankyrin domain alone was sufficient to up-regulate some, but not all, RBP-Jkappa-dependent genes. Although N4IC reduced VEGF receptor-2 (VEGFR-2) and vascular endothelial (VE)-cadherin expression, neither of these events is necessary and sufficient to explain N4IC-mediated inhibition of sprouting. A constitutively active RBP-Jkappa mutant significantly inhibited HMEC sprouting but not as strongly as N4IC. Thus, Notch4-induced inhibition of sprouting requires the ankyrin repeats and appears to involve RBP-Jkappa-dependent and -independent signaling.

Notch activation results in phenotypic and functional changes consistent with endothelial-to-mesenchymal transformation.

Various studies have identified a critical role for Notch signaling in cardiovascular development. In this and other systems, Notch receptors and ligands are expressed in regions that undergo epithelial-to-mesenchymal transformation. However, there is no direct evidence that Notch activation can induce mesenchymal transdifferentiation. In this study we show that Notch activation in endothelial cells results in morphological, phenotypic, and functional changes consistent with mesenchymal transformation. These changes include downregulation of endothelial markers (vascular endothelial [VE]-cadherin, Tie1, Tie2, platelet-endothelial cell adhesion molecule-1, and endothelial NO synthase), upregulation of mesenchymal markers (alpha-smooth muscle actin, fibronectin, and platelet-derived growth factor receptors), and migration toward platelet-derived growth factor-BB. Notch-induced endothelial-to-mesenchymal transformation does not seem to require external regulation and is restricted to cells expressing activated Notch. Jagged1 stimulation of endothelial cells induces a similar mesenchymal transformation, and Jagged1, Notch1, and Notch4 are expressed in the ventricular outflow tract during stages of endocardial cushion formation. This is the first evidence that Jagged1-Notch interactions induce endothelial-to-mesenchymal transformation, and our findings suggest that Notch signaling may be required for proper endocardial cushion differentiation and/or vascular smooth muscle cell development.

Bacterial lipopolysaccharide directly induces angiogenesis through TRAF6-mediated activation of NF-kappaB and c-Jun N-terminal kinase.

The intracellular pathways by which inflammatory mediators transmit their angiogenic signals is not well studied. The effects of a potent inflammatory mediator, bacterial lipopolysaccharide (LPS), are transmitted through Toll-like receptors (TLRs). A major, although not exclusive, LPS/TLR intracellular signaling pathway is routed through TNF (tumor necrosis factor) receptor associated factor 6 (TRAF6). In this report we demonstrate that LPS directly stimulates endothelial sprouting in vitro. By blocking TRAF6 activity using retroviral expression of a dominant-negative TRAF6 in endothelial cells, we show that TRAF6 is absolutely required for the LPS-initiated angiogenic response in vitro and in vivo. Inhibition of either c-Jun N-terminal kinase (JNK) activity or nuclear factor kappaB (NF-kappaB) activity, downstream of TRAF6, is sufficient to inhibit LPS-induced endothelial sprouting. In contrast, only inhibition of NF-kappaB, but not JNK, activity blocks basic fibroblast growth factor (bFGF)-induced angiogenesis. Our findings thus demonstrate a direct endothelial-stimulatory role of LPS in initiating angiogenesis through activation of TRAF6-dependent signaling pathways.

Vascular endothelial growth factor receptor-2 induces survival of hematopoietic progenitor cells.

Vascular endothelial growth factor (VEGF) and its receptors play an essential role in the formation and maintenance of the hematopoietic and vascular compartments. The VEGF receptor-2 (VEGFR-2) is expressed on a population of hematopoietic cells, although its role in hematopoiesis is still unclear. In this report, we have utilized a strategy to selectively activate VEGFR-2 and study its effects in primary bone marrow cells. We found that VEGFR-2 can maintain the hematopoietic progenitor population in mouse bone marrow cultured in the absence of exogenous cytokines. Maintenance of the hematopoietic progenitor population is due to increased cell survival with minimal effect on proliferation. Progenitor survival is mainly mediated by activation of the phosphatidylinositol 3'-kinase/Akt pathway. Although VEGFR-2 also activated Erk1/2 mitogen-activated protein kinase, it did not induce cell proliferation, and blockade of this pathway only partially decreased VEGFR-2-mediated survival of hematopoietic progenitors. Thus, the role of VEGFR-2 in hematopoiesis is likely to maintain survival of hematopoietic progenitors through the activation of antiapoptotic pathways.