La transfusion sanguine est une thérapeutique indispensable pour laquelle il n’existe pas actuellement de substitut. La transfusion de produits sanguins labiles est dans la grande majorité des cas très bien tolérée mais elle peut être à l’origine d’effets indésirables chez les receveurs (EIR) notamment de type inflammatoire. Ceci dépend de facteurs liés aux produits eux-mêmes et/ou aux receveurs de par leur prédisposition génétique et de leur état clinique. Les concentrés plaquettaires (CP) sont la principale source de manifestations inflammatoires et/ou allergiques. Ceci est notamment dû, en partie, à la capacité des plaquettes à sécréter une multitude de molécules ayant une activité inflammatoire. De plus, les processus de collecte, de préparation et de conservation induisent un stress vis-à-vis des cellules, qui peut activer les plaquettes et donc induire la production de produits inflammatoires dans les CP. Le but de ce travail de thèse a été dans un premier temps d’identifier les molécules les plus impliquées dans les manifestations inflammatoires. Le sCD40L en particulier est identifié comme étant largement impliqué dans les EIR après transfusion de CP, mais pas systématiquement. Aussi, la composante inflammatoire de ces réactions est multifactorielle. De plus, nous avons évalué le potentiel inflammatoire des CP sur l’endothélium vasculaire. Des différences d’activation des cellules endothéliales, dans un modèle in vitro, ont été observées lorsqu’elles sont en présence de surnageants de CP ayant induits un EIR. Ce travail de thèse poursuit l’effort entrepris par notre équipe de recherche, en vue de prédire la survenue d’EIR et de préciser les mécanismes qui influencent la physiopathologie plaquettaire transfusionnelle ; un corollaire de ces travaux est ainsi d’optimiser les processus de production et de conditionnement des CP transfusés afin de réduire ces réactions inflammatoires. / Blood transfusion is an indispensable therapy for which there is currently no substitute. Transfusion of blood products is in the great majority of cases very well tolerated but it can be at the origin of serious adverse reactions (SARs), notably of inflammatory reactions. This depends on the factors related to the products themselves and/or to the recipients, their genetic predisposition and clinical condition. Platelet concentrates (PCs) are the main source of inflammatory and/or allergic manifestations. This is due, in part, to the ability of platelets to secrete a multitude of molecules with inflammatory activity. In addition, the collection, processing and storage conditions induce stress on cells, which can activate platelets and thus induce the production of inflammatory products in PCs. The purpose of this work is to identify the molecules involved in inflammatory manifestations. sCD40L was identified as being involved in SARs after PCs transfusion, but not systematically. Also, the inflammatory component of these reactions is multifactorial. In addition, we evaluated the inflammatory potential of PCs on the vascular endothelium. Differences in endothelial cell activation, in an in vitro model, were observed when they were in the presence of PC supernatants involved in SARs. This thesis work continues the effort undertaken by our research team to predict the occurrence of SARs and to clarify the mechanisms that influence transfusional platelet physiopathology; a corollary of this work is to optimize the production and conditioning process of PCs transfused in order to reduce these inflammatory reactions.

The incidences of obesity and type 2 diabetes and their complications are increasing globally. The presence of elevated circulating free fatty acids has been associated with the initial dysfunction of pancreatic beta cells and microvascular endothelial cells followed later by their demise. The aim of this thesis was to investigate the mechanisms by which demise occurs, and how it may be prevented. Palmitate, a saturated fatty acid, caused cell death in both INS-1 beta cells and HCMec/D3 microvascular cells, whereas the unsaturated fatty acid oleic acid did not cause cell death, and also protected against palmitate-induced toxicity. Etomoxir, the mitochondrial CPT1 inhibitor did not rescue INS-1 or HCMec/D3 cells from palmitate-induced toxicity suggesting that palmitate-induced toxicity does not occur via entry into the mitochondria. Cells were exposed to 2-bromopalmitate, a non-metabolisable fatty acid used to reduce the pool of cytoplasmic CoA, to determine whether palmitate-induced toxicity might be mediated by its ability to be activated. Pre-incubation with 2-bromopalmitate in INS-1 cells significantly prevented palmitate-induced cell death. These data suggest that the activation of palmitate with CoA might mediate cell death. Cell cycle analysis found that neither oleic acid nor palmitate caused an increase or decrease in cell proliferation in both INS-1 and HCMec/D3 cells. The data suggest that the mechanism of oleic acid-induced cytoprotection might not be via a pro-proliferative mechanism. INS-1 cells were imaged using spontaneous Raman microspectroscopy after 24-hour exposure to esterified and non-esterified fatty acids. Uni- and multi-variate analysis and spectral decomposition were carried out using a methodology optimised and validated which is presented in this thesis. The aim was to quantify changes, if any, in lipid disposition: distribution, intensity (as a measure of concentration) and composition after exogenous exposure to these fatty acids. Exposure to 0.125 mM palmitate showed a significant decrease in the percentage of lipid within the cells and a corresponding increase in the intensity of this lipid. This suggests that palmitate, alone, might be shuttled into lipid droplets. This was not observed when the cells were exposed to oleic acid, whereby an increase in the intensity of lipid was observed even though no significant change was observed in the percentage of lipid within the cells. When palmitate and oleic acid were combined, the composition of the lipid droplets changed such that the levels of palmitate decreased and the levels of oleic acid increased. These data suggest that oleic acid does not shuttle palmitate into lipid droplets. These data do not support the hypothesis that oleic acid protects against palmitate-induced cytotoxicity by shuttling palmitate into lipid droplets. The methyl esters of palmitate and oleic acid were employed to determine whether they would affect lipid disposition. No change in lipid distribution or intensity was observed when the cells were exposed to these fatty acids, validating the requirement for the free carboxyl oxygen for the covalent binding to glycerol for the formation of lipid droplets. These data also suggest that INS-1 cells cannot de-esterify esterified fatty acids.

Orientador: Willian Fernando Zambuzzi / Resumo: Mudanças nas forças tensionais do shear-stress estão associadas a um repertório de cascatas de sinalização celular, as quais modulam em conjunto o fenótipo vascular tornando o tecido endotelial susceptível a variações patofisiológica e, portanto, compreensão do repertório molecular neste cenário é necessária. Com este propósito, nós submetemos células endoteliais de veia umbilical humana (HUVEC) a um circuito de diferentes forças tensionais in vitro, considerando os grupos seguintes: 1. condição de fluxo de shear-stress fisiológico (nomeado Normo); 2. fluxo de shear-stress hipertenso (nomeado Hyper), e 3. células do grupo 2 foram retornadas para a condição Normo (nomeado Return). As amostras foram apropriadamente coletadas para seguir em diferentes metodologias. Nossos resultados mostraram um forte envolvimento de c-Src no controle da cascata de mecanotransdução modulando sinalização necessária para o fenótipo de adesão, sobrevivência (PI3K/AKT) e proliferação celulares. Além disso, c-Src parece desenvolver importante papel durante o remodelamento da Matriz Extracelular (MEC), cujo performance de matriz metaloproteinases (MMPs) mostrou mudanças significativas. Além disso, através de análise proteômica, mostramos um forte envolvimento de Heat Shock Protein 70 (HSP70) nas células estressadas de modo Hyper, reduzindo significativamente no grupo Return. Esse resultado levou-nos a investigar o proteassoma 20S como uma alternativa proteolítica intracelular para promover o turnover ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Shear-stress changes are associated with a repertory of signaling cascade, modulating vascular phenotype. As shear stress-related tensional forces might be associated with pathophysiological susceptibility, a more comprehensive molecular map needs to be addressed. Thus, we subjected human umbilical vein endothelial cells (HUVECs) to a circuit of different tensional forces in vitro considering the following three groups: one in a physiological blood flow shear-stress condition (named Normo), another in which these cells followed to a hypertensive blood flow shear-stress (named Hyper), and finally one that these hyper-stressed cells were returned to Normo condition (named Return). The samples were properly collected to allow different methodologies analysis. Our data showed a pivotal involvement of c-Src on driving the mechanotransduction cascade by modulating signaling related with adhesion, survival (PI3K/Akt) and proliferative phenotype. Moreover, c-Src seems to develop important role during Extracellular Matrix (ECM) remodeling, which showed significative changes. Additionally, proteomic analysis showed strong involvement of Heat Shock Protein 70 (HSP70) in the hypertensive-stressed cells; it being significantly decreased in Return phenotype. This result prompted us to investigate 20S proteasome as an intracellular proteolytic alternative to promote the turnover of those proteins. Surprisingly, our data reveled significant over expression of sets of proteasome subunit α-typ... (Complete abstract click electronic access below) / Mestre

Moléculas de origem vegetal foram há muito testadas como fonte de drogas antineoplásicas com sucesso promissor. Este trabalho trata dos efeitos antiangiogênicos do Metil Jasmonato. Este derivado hidrofóbico do ácido jasmônico foi demonstrado anteriormente como um agente de dano seletivo para a mitocôndria de células neoplásicas. In vitro, o Metil Jasmonato 1-10 mM promoveu a morte celular de células endoteliais humanas de cordão umbilical (HUVEC) e de melanoma murino (B16 -F10), enquanto concentrações micromolares foram inócuas. A inclusão do Metil Jasmonato em liposomos de fosfatidilcolina e em um nanocarreador hidrofílico baseado em açúcar mostrou efeitos diferenciais sobre a citotoxicidade. A interrupção do ciclo celular foi observada em concentrações citotóxicas, enquanto a diminuição na produção de VEGF e algum grau de autofagia foram sugeridos em concentrações micromolares. In vivo, Metil Jasmonato 1-10mM foi francamente tóxico, e reduziu a densidade de vasos em membranas corioalantóicas de embrião de galinha (CAM). Entretanto, concentrações entre 1-10 ?M produziram um efeito complexo. Ocorreu aumento no brotamento capilar, mas os novos vasos apresentaram-se frágeis e menos organizados que os controles correspondentes. Sugere-se que, além da toxicidade direta, a ação do Metil Jasmonato sobre a angiogênese seja relevante para seu efeito antineoplásico. / Molecular plant components have long been tested as sources for antineoplasic drugs with promising success. The present work deals with the anti-angiogenic effects of Methyl Jasmonate. This hydrophobic Jasmonate derivative was previously demonstrated to selectively damage the mitochondria of cancer cells. In vitro, 1-10 mM Methyl Jasmonate induced the cell death of the human umbilical vein endothelial cells (HUVEC) and the Murine melanoma cells (B16-F10), while micromolar concentrations were ineffective. Methyl Jasmonate inclusion in phosphatidylcholine liposomes and in an hydrophilic sugar based nanocarrier presented differential effects upon citotoxicity. Cell cycle arrest was observed in citotoxic concentrations, while VEGF withdrawn and some autophagy was suggested in the micromolar range. In vivo, 1-10mM concentrations were explicitly toxic and reduced the vessel density of the Chorioallantoic Membrane of the Chicken Embryo (CAM). However, 1-10 ?M concentrations produced a complex effect. There was increased capillary budding, but the new vessels were leakier and less organized than corresponding controls. It is suggested that not only direct toxicity, but also the drug effects upon angiogenesis are relevant to the antineoplasic effects of Methyl Jasmonate.

Chronic kidney disease (CKD) is growing global public health problem affecting 1 in 10 adults in developed countries and recognised as an important risk factor for cardiovascular disease (CVD) development. CVD is the main cause of death among CKD patients. Endothelial injury and dysfunction are critical steps in atherosclerosis, a major CVD. Oxidative stress (increased level of reactive oxygen species, ROS) has been associated with CVD development. Intravenous (IV) iron preparations are widely used in the management of CKD mediated anaemia, and have been associated with increased oxidative stress and cellular dysfunction. This study examined the effect of pharmacologically-relevant concentrations of IV Venofer (iron sucrose) or IV Ferinject (Ferric carboxymaltose, FCM) on primary human umbilical vein endothelial cell (HUVEC) activation/damage and on intracellular ROS generation as well as studying the potential mechanisms responsible. Data from TUNEL assay and Annexin V-FITC/PI staining showed that, IV FCM had no effect, but IV iron sucrose increased HUVEC apoptosis at 24hr. IV iron sucrose inhibited cell proliferation and reduced cell viability. Both compounds induced EC activation through sustained activation of p38 MAPK and up-regulation of ICAM-1 and VCAM-1. Additionally, the compounds induced significant increase in total ROS and superoxide anion production, which was attenuated by the anti-oxidant N-acetylcysteine (NAC). P38 MAPK showed up-regulation of pro-apoptotic protein Bax and down-regulation of antiapoptotic Bcl-2 protein in HUVEC treated with IV iron sucrose and p38 inhibition reversed these effects. In summary, these results suggest that IV iron sucrose causes more severe EC injury than IV FCM. However, both IV iron preparations induced intracellular ROS and superoxide anion generation in HUVEC leading to EC activation/dysfunction, providing a potential explanation for vascular damage in CKD patients.

The potential of embryonic stem (ES) cells to generate any developmental or adult cell type holds much promise for regenerative medicine and in vitro modelling of development and disease. Haematopoietic stem cells (HSCs) regenerate all lineages of the blood throughout adult life and are essential for the treatment of a vast number of haematalogic disorders. Current sources of HSCs for clinical use and research, including adult bone marrow, peripheral blood stem cells and umbilical cord blood, are limited by the number of HSCs they contain and by the availability of a suitable donor. A system that generates a reliable source of HSCs from ES cells would therefore be an ideal alternative. While much progress has been made in the generation of downstream lineages of the haematopoietic system, progress in the derivation of HSCs capable of long-term self-renewal and multilineage reconstitution from ES cells has been limited. Understanding of the developmental steps leading to HSC emergence in the embryo has been advancing in recent years. In particular, precursors of HSCs (preHSCs) have been isolated from the mouse embryo, characterised and matured into HSCs ex vivo using the specialised conditions of aggregate culture systems (Taoudi et al 2008, Rybtsov et al 2011). We hypothesised that application of the aggregate culture system in the differentiation of ES cells could provide a missing link in the in vitro generation of HSCs. Here I have developed a novel ES cell differentiation system that employs the specialised conditions of the aggregate culture system, after an initial stage of mesoderm differentiation. I show that this system creates an environment for efficient haematopoietic and endothelial progenitor formation and generates cells of a preHSC type I (VE-Cadherin+CD45-CD41lo) and preHSC type II (VE-Cadhein+CD45+) surface phenotype. Notably, the system gives rise to cells that achieve low-levels of haematopoietic repopulation in sublethally irradiated NSG mice. The low-level repopulating cells persist for over 4 months in animals and show both myeloid and lymphoid potential. I identify genes that are expressed in cells of a preHSC II surface marker-phenotype from the E11.5 dorsal aorta, but not in cells of this phenotype from the E11.5 Yolk sac or differentiated ES cells. I also show that enforced expression of Notch downstream target Hes1 in Flk1+ mesoderm during ES cell differentiation does not improve levels of ES-derived repopulation.

Development of an in vitro model for the early stages of cardiovascular disease is a current necessity. Cardiovascular disease is the leading cause of death in the United States and throughout the world. Oxidative stress and reactive oxygen species have been implicated in cardiovascular disease development. An in vitro model of these processes will improve our understanding of cardiovascular disease development and allow for the development of additional treatments. Atherosclerosis is an inflammatory disease and increased levels of H2O2 are associated with inflammation. The model focuses on H2O2-induced oxidative stress under static and shear conditions. Previous studies have documented increased O2.- and increased cytotoxicity in smooth muscle cells exposed to H2O2. Under static culture, endothelial cells exposed to H2O2, exhibited increased O2.- over basal levels via NOS and NAPDH oxidase pathways. Increased O2.- was attenuated by MnSOD adenoviral-mediated upregulation and endothelial cell exposure to Tiron. This suggests NOS and NADPH oxidase as sources of increased O2.- under H2O2-induced oxidative stress. Endothelial cell cytotoxicity was increased with H2O2 exposure. The increase in cytotoxicity was diminished upon exposure to Tiron or L-NAME. Under shear conditions (8.2 dynes/cm2), endothelial cells exposed to H2O2 exhibited increased O2.- compared to control via an L-NAME (specific inhibitor NOS) and Apocynin (NADPH oxidase inhibitor) inhibitable mechanism. This suggests NOS and NADPH oxidase as sources of increased O2.- under H2O2-induced oxidative stress. The increased O2.- was attenuated with MnSOD adenoviral-mediated upregulation and endothelial cell exposure to Tiron (an O2.-scavenger). Endothelial cell attachment under shear with exposure to H2O2 was improved with MnSOD adenoviral-mediated upregulation as observed by decreased loss of the endothelial cell monolayer compared with H2O2 exposed endothelial cells. Endothelial cells exposed to H2O2 exhibit increased O2.-, suggesting that H2O2-induced oxidative stress may be a reasonable model for atherosclerosis. NOS and NADPH oxidase co-inhibition under shear and static culture demonstrated that NOS and NADPH oxidase inhibition is non-additive under static culture, yet additive under shear. Co-inhibition results suggest a complex relationship between the two enzymes that requires additional experimentation to deconvolve.

<p>The mechanisms behind many important aspects of blood- and lymphatic vessel formation have yet not been elucidated in detail. The primary objectives of this thesis have therefore been to study the effects of hypoxia, platelet-derived growth factor (PDGF) and vascular endothelial growth factors (VEGFs) on vascular development and function. </p><p>In conditions of low oxygen pressure, hypoxia, the survival of the organism is critically dependent on the ability to compensate for the reduced oxygen levels by promoting blood vessel growth and oxygen-independent energy production. Many direct effects of hypoxia in cells are attributed to the induction of a family of hypoxia-inducible transcription factors (HIFs) which control the expression of specific target genes. We found that capillary endothelial cells (ECs) respond to hypoxia with upregulation of genes involved in growth and remodeling of blood vessels. On the other hand, vein ECs responded to hypoxia with increased expression of genes involved in lymphatic vessel growth. Using differentiating embryonic stem (ES) cells, we have shown that hypoxia upregulates expression of VEGF receptor-3 (VEGFR-3) on blood vascular ECs. Furthermore, we have provided evidence for a critical role of VEGFR-3 in hypoxia-induced blood vessel development. </p><p>Activation of PDGF receptor-β (PDGFR-β) on early vascular progenitors in differentiating ES cells or in mice induces blood vessel differentiation, while negatively influencing early hematopoiesis. PDGFR-β expression on vascular progenitors may therefore play a role in guiding differentiation of the vascular lineages. </p><p>We have investigated the usefulness of differentiating ES cells as a model to study early lymphatic development. Administration of VEGF-C and VEGF-A induced formation of lymphatic vessel-like structures that seemed connected to the blood vasculature, supporting the general view that lymphatic ECs are derived from blood vascular ECs.</p><p>In summary, this thesis has provided new insights in the contribution of different growth factors in hematopoietic, blood- and lymphendothelial development. </p>

Substanzen der pharmazeutischen und chemischen Industrie müssen nach internationalen Richtlinien auf deren Toxizität gegenüber Mensch und Umwelt geprüft werden. Dazu gehören u. a. Prüfungen zur Vorhersage des embryotoxischen Potentials, die am lebenden Organismus durchgeführt werden. Mit dem Ziel die Anzahl der Tierversuche zu verringern, die notwendig sind um das toxikologische Profil einer Prüfsubstanz zu bestimmen, wurde der Embryonale Stammzelltest (EST) entwickelt. Als Grundlage des EST dienen embryonale Stammzellen (ES-Zellen) einer Zelllinie. ES-Zellen sind Zellen, die sich in der frühen embryonalen Entwicklung in die Zellen der Keimblätter entwickeln können. Daraus wiederum differenzieren die vielen verschiedenen, unterschiedlich spezialisierten Zelltypen des komplexen Organismus. Im EST wird die Konzentration einer Prüfsubstanz bestimmt, bei der die Differenzierung von ES-Zellen zu Herzmuskelzellen zu 50 % inhibiert wird. Zusätzlich wird die Konzentration der Prüfsubstanz bestimm&#176;t, bei der 50 % der ES-Zellen (IC50D3) bzw. Fibroblastenzellen (IC503T3) absterben. Die allgemeine Toxizität ist damit von der spezifischen Toxizität der Prüfsubstanz auf die ES-Zellen und deren Differenzierung unterscheidbar. Die Parameter fliessen in ein biostatistisches Modell zur Prädiktion des embryotoxischen Potentials der Prüfsubstanzen ein. <br><br> Es wurde ein Versuchsprotokoll entwickelt, wonach die ES-Zellen sich verstärkt zu Endothelzellen differenzieren. Die Endothelzellen, die im lebenden Organismus die Wand der späteren Blutgefässe, wie Venen und Arterien bilden, wurden mittels molekularbiologischer Methoden auf der RNA- und der Protein-Ebene nachgewiesen und quantifiziert. Verschiedene Zellkulturmethoden, Wachstumsfaktoren, als auch Wachstumsfaktorkonzentrationen wurden auf deren Vermögen die Differenzierung der ES-Zellen zu Endothelzellen zu induzieren, untersucht. Nach der Etablierung des Differenzierungsprotokolls wurden sieben Substanzen auf deren Vermögen geprüft, die Differenzierung von ES-Zellen zu Endothelzellen zu inhibieren. Die Endothelzellen wurden dabei über die Expression der RNA von zwei endothelzellspezifischen Genen quantifiziert. Im Vergleich dazu wurden die IC50D3 und die IC503T3 der Prüfsubstanz bestimmt, um eine Abschätzung des embryotoxischen Potentials der Prüfsubstanz zu ermöglichen. Die Ergebnisse zeigten, dass eine Abschätzung des embryotoxischen Potentials der sieben Prüfsubstanzen in nicht-, schwach- oder stark embryotoxisch vorgenommen werden konnte. Es ist zu schlussfolgern, dass der weiterentwickelte in vitro Embryotoxizitätsassay sensitiv und reproduzierbar ist. Mit der Verwendung von verschiedenen Differenzierungsendpunkten kann die Prädiktionskraft des Assays deutlich verbessert, und die Anzahl von Tierversuchen verringert werden. Durch die Verwendung von molekularbiologischen Markern kann der Assay einem Hochdurchsatzscreening zugängig gemacht werden und damit die Anzahl von Prüfsubstanzen deutlich erhöht werden. / Compounds of the pharmaceutical and chemical industry need to be tested for their toxicological potential with regard to humans and environment following international guidelines. Tests for the prediction of the embryotoxic potential executed on living organisms are examples of these guidelines. In order to reduce the number of animal experiments necessary for the assessment of the toxicological profile of compounds the embryonic stem cell test (EST) was developed. Embryonic stem cells (ES-cells) of a cell line are used as the basis of the EST. ES-cells are cells which develop at the early embryonic development into cells of the germ layers. Out of these the many different specialized cell types of the complex organism can differentiate. With the EST this concentration of a test compound will be determined where a 50 % inhibition of the differentiation of ES-cells into cardiomyocytes can be detected. Additionally, the concentration of a test compound which is cytotoxic to 50 % of the ES-cells (IC50D3) and to 50 % of fibroblasts (IC503T3) will be determined. Therefore, general toxicity caused by the test compound on ES-cells and its differentiation can be distinguished from specific toxicity of the test compound. Determined parameters will be included into a biostatistical model for the subsequent predicition of the embryotoxic potential of test compounds. <br><br> A protocol was developed whereby the differentiation of ES-cells into endothelial cells is induced. Endothelial cells which make up the walls of blood vessels, such as arteries and veins, were detected and quantified at the RNA and the protein level applying molecular biological methods. Different cell culture methods, growth factors and growth factor concentrations were studied for their ability to induce the differentiation of ES-cells into endothelial cells. Applying the developed differentiation protocol seven compounds were tested for their potential to inhibit the differentiation of ES-cells into endothelial cells. Endothelial cells were quantified by the RNA-expression of two endothelial-specific genes. In comparison to the expression levels the IC50D3 and the IC503T3 were determined in order to assess the embryotoxic potential of the test compound. The results showed that an assessment of the embryotoxic potential of the seven test compounds into non-, weakly- and strongly embroytoxic was possible. It can be concluded that the improved in vitro embryotoxicity assay is sensitive and reproducible. With the use of different differentiation endpoints the power of predicitivity of this assay can be significantly increased and the number of animal experiments can be reduced. With the application of molecular biological markers this assay can be applied as a high througput screening and therefore the number of test compounds can be strongly increased.

Delta-like 1 Homologue (DLK1), a transmembrane protein of 383 amino acids, belongs to a family of epidermal growth factor (EGF)-like repeat-containing proteins that include Notch/Delta/Serrate, which are involved in cell fate determination. DLK1 is also known as preadipocyte factor-1, pG2, and FA-1, which are identical or polymorphic products of a single gene. Structural analysis revealed that DLK1 consists of an extracellular domain with six EGF-like repeats, a transmembrane domain, and an intracellular domain. The extracellular EGF-like region of DLK1 (DLK1-EC) can be released to the medium by the action of tumor necrosis factor alpha converting enzyme (TACE). DLK1 participates in various differentiation processes including adipogenesis, hematopoiesis, and adrenal gland differentiation. Besides, DLK1 overexpression was observed in patients with biliary atresia and in glioblastoma. Recently, the extracellular domain of thrombomodulin, which also contains six EGF¡Vlike structures, has been delineated to stimulate angiogenesis in vitro and in vivo. This prompted us to investigate whether DLK1-EC played a role in angiogenesis. To test such hypothesis, recombinant DLK1-EC was expressed and purified in E. coli. Adding DLK1-EC recombinant protein inhibited the adipogenesis of adipocytes-derived stem cells in a dose-dependent manner. Despite marginal effect on matrix-metalloproteinase secretion, exogenous DLK1-EC significantly stimulated the proliferation, motility and tube-forming capability of cultured endothelial cells. Above all, implantation of DLK1-EC-containing hydron pellets induced cornea neovascularization in a dose-dependent manner. Western blot analysis revealed that exogenous DLK1-EC induced angiogenesis through Notch1 activating downstream gene Hes1 and subsequently signaling such as Akt/eNOS, p38 MAPK, and ERK pathway to perform its function. Indeed, blockade of Notch1 signaling using £^-secretase inhibitor leads to decreased angiogenesis and inhibits DLK1 EC-induced endothelial cell tubular formation in vitro and in vivo. These findings indicate that DLK1-EC induced Notch1 activation mediated by £^-secretase and tansactivation Akt/eNOS pathway and that Notch1 is critical for DLK1 EC-induced angiogenesis. These results may bring further insights into the physiological and pathological functions of DLK1