published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

Histidine-rich glycoprotein (HRG) is a plasma protein that regulates key cardiovascular processes such as coagulation, angiogenesis and immune response. The protein consists of six distinct functional domains: two N-terminal domains (N1 and N2), two proline-rich regions (PRR1 and PRR2), a central histidine-rich region (HRR) and a C-terminal domain. The HRR binds Zn²⁺, which alters the affinity of HRG towards various ligands including the anticoagulant, heparin. A key aim of this study was to structurally characterise HRG. The 1.93 Å crystal structure of the HRG N2 domain presented here represents the first crystallographic snapshot of the molecule. The N2 domain is cystatin-like and N-glycosylated at Asn184. An S-glutathionyl adduct was observed at Cys185, providing in vivo evidence that release of an anti-angiogenic HRR/PRR fragment is controlled in part by a redox mechanism, representing a novel further role for GSH in regulation of angiogenesis. Since Zn²⁺ regulates some of the functions of HRG, the dynamics of Zn²⁺ in plasma were investigated using a combination of ITC, ELISA and thrombin assay systems. Zn²⁺ is normally associated with albumin in circulation, but its ability to bind Zn²⁺ is allosterically inhibited upon fatty acids binding to albumin. Elevated plasma fatty acid levels are associated with some disease states. It is proposed that this may alter the proportion of Zn²⁺ bound to HRG, which could in turn activate thrombin to promote coagulation. These studies provide evidence to suggest that Zn²⁺-dependent activation of HRG (following fatty acid binding to albumin) may play a role in the development of haemostatic complications in susceptible individuals. Finally, the Zn²⁺ binding ability of albumin was probed in order to locate unidentified sites using recombinant albumin mutants. H9A, H67A, E252A, D256A and H288A mutants all exhibited diminished Zn²⁺ binding ability, indicating that these residues are involved directly or indirectly in Zn²⁺ binding.

Orientador: Andrigo Barboza De Nardi / Banca: Paola Castro Moraes / Banca: Carlos Henrique Maciel Brunner / Resumo: O carcinoma de células escamosas (CCE) é a segunda neoplasia maligna cutânea que mais acomete os cães. Apesar da cirurgia consistir na terapia de eleição, a eletroquimioterapia (EQT) vem sendo cada vez mais utilizada. O objetivo desta pesquisa foi avaliar a resposta tecidual em CCE cutâneo em cães após 21 dias da EQT. Desta maneira, este é o primeiro estudo a avaliar as modificações nos fatores angiogênicos e reparação tecidual induzidas pela EQT em CCE cutâneo de em cães. Foram avaliados 11 cães, totalizando-se 18 lesões, antes da EQT (D0) e 21 dias após (D21). O protocolo de EQT consistiu na aplicação de sulfato de bleomicina seguida por cloridrato de doxorrubicina utilizando eletroporador LC BK-100. Foram determinados volume tumoral, índice mitótico, necrose microscópica, quantificação das fibras colágenas do tecido conjuntivo (pela coloração de Tricrômico de Masson) e a expressão imuno-histoquímica de fatores angiogênicos VEGF e CD31. Observou-se que a média do volume tumoral foi significativamente inferior ao 21º dia (D21: 21,99±37,55) após a realização da EQT (D0: 29,52±40,95) (p=0,044). Contudo, não foram encontradas diferenças para as demais variáveis (p>0,05). Conclui-se que, em CEC de cães, a EQT não foi capaz de alterar o índice mitótico, a necrose microscópica e as estruturas de colágeno, além dos fatores angiogênicos VEGF e CD31, após 21 dias do tratamento. Apesar disso, no mesmo momento de avaliação, redução significativa do volume tumoral pode ser observada. Su... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Squamous cell carcinoma (SCC) is the second most common cutaneous malignant neoplasm in dogs. Electrochemotherapy (ECT) is a recent technique in veterinary medicine that constitutes an alternative and effective form to treat SCC in dogs and cats. The aim of this study was to evaluate modifications in the angiogenic factors and tissue repair induced by ECT after 21 days of ECT. Eleven dogs were evaluated (18 lesions), before (D0) and after (D21) ECT. The ECT protocol consisted of the application of bleomycin sulfate followed by doxorubicin hydrochloride using the BK-100 LC electroporator. Tumor volume, mitotic index, microscopic necrosis, quantification of connective tissue collagen fibers (by Masson's trichrome staining) and immunohistochemical expression of angiogenic factors by VEGF and CD31 were determined. It was observed a significant reduction in size of the tumor volume after ECT (D0: 29.52 ± 40.95; D21: 21.99 ± 37.55) (p = 0.044). However, no differences were found in D0 and D21 for mitotic index, microscopic necrosis, collagen structures and angiogenic factors (p> 0.05). In conclusion, ECT is an effective technique to treat cutaneous SCC in dogs, however cell proliferation, necrosis and angiogenic factors were not affected after 21 days of ECT, suggesting a prior moment to be evaluated. Further studies on other angiogenic markers and tissue repair in cutaneous SCCs of dogs submitted to ECT are necessary. / Mestre

Tumores são definidos como microambientes compostos não só pelas células malignas, mas também por células endoteliais, fibroblastos e leucócitos, que promovem o crescimento tumoral e a angiogênese. Galectina-3, uma proteína que se liga a b- galactosídeos, é abundantemente expressa por monócitos/macrófagos, dentre outros leucócitos. Inúmeras evidências sugerem que galectina-3 atua como uma molécula reguladora da resposta inflamatória. Tendo em vista que o infiltrado inflamatório pode promover a progressão de tumores, o objetivo do presente trabalho foi avaliar se galectina-3, expressa tanto pela célula tumoral como pelas células estromais, modula o crescimento de melanoma. Para tal, células de melanoma murino Tm1 foram transfectadas com o gene de galectina-3. Ambos clones celulares (galectina-3 positivos e negativos) foram injetados na intrafáscia ou no subcutâneo de camundongos (fêmeas) C57BL/6 selvagens e/ou nocautes para o gene de galectina-3 para análise da implantabilidade e crescimento tumoral. Com relação à implantabilidade, não foi observado diferenças no estabelecimento de uma massa tumoral proliferativa em animais selvagens inoculados com células Tm1 transfectadas ou não com o gene de galectina-3 em animais selvagens. Em relação a taxa de crescimento dos tumores, nenhum animal nocaute inoculado com células Tm1 galectina-3 positivas apresentou tumores de dimensões mensuráveis até o 11º dia pós-inóculo. Independente do nível de expressão de galectina- 3 pela célula tumoral, os tumores originados nos animais nocautes apresentavam menor massa em gramas comparados ao grupo selvagem, sugerindo que galectina-3 expressa pelas células estromais promove o crescimento tumoral. Ainda, os tumores originados nos animais nocautes e no grupo selvagem inoculado com células Tm1 galectina-3 positivas apresentavam menor extensão de área necrótica do que os animais selvagens inoculados com células Tm1 galectina-3 negativas. Interessantemente, os animais selvagens e nocautes inoculados com células Tm1 galectina-3 positivas apresentaram tumores com menor área vascular e menor número de estruturas vasculares funcionais quando comparados aos animais selvagens inoculados com células Tm1 galectina-3 negativas. A análise de expressão gênica nos tumores mostrou que os níveis relativos de RNAm de VEFG (fator de crescimento de endotélio vascular) foram menores nos animais inoculados com células Tm1 galectina-3 positivas em relação aos inoculados com células Tm1 galectina-3 negativas, indicando que galectina-3 expressa pelas células tumorais atua como uma molécula anti-angiogênica. Finalizando, o presente trabalho sugere que galectina-3 pode atuar como uma molécula pró- ou anti-tumoral, dependendo do tipo celular que a expressa no microambiente tumoral. / Tumors have been described as microenvironments composed not only by malignant cells, but also by endothelial cells, fibroblasts and leukocytes, which can promote tumor growth and angiogenesis. Galectin-3, a b-galactoside binding protein, is expressed by monocytes/macrophages and others leukocytes. In fact, several lines of evidence suggest that galectin-3 act as master regulators of the inflammatory response. Based on the fact that the inflammatory infiltrate can promote tumor progression, the proposal of this study was to evaluate if galectin-3, either from tumor or stromal cells could modulate melanoma growth. Tm1 murine melanoma cell line was transfected with the galectin-3 gene. Both clones (galectin-3 negative and positive) were injected in the foot pad or subcutaneous in female C57BL/6 wild-type (WT) and galectin-3 knock-out (KO) mice to tumor engraftment and growth analysis. There was no difference in the tumor engraftment between animas injected with Tm1 galectin-3 positive or negative cells. In addition, any knock-out mice injected with galectin-3 positive cells had measurable tumors up to day 11 post inoculation. Regardless the galectin-3 expression level in the melanoma cell, tumors from galectin-3 KO mice were smaller than those from WT animals, suggesting that galectin-3 expressed by stromal cells promotes tumor growth. Moreover, tumor necrotic area was smaller in KO mice and in wild-type animals injected with Tm1 galectin-3 positive cells compared to wild type animals injected with Tm1 galectin-3 negative cells. Interestingly, both vascular area and the number of functional vessels in animals injected with galectin-3 positive Tm1 cells were smaller in WT as well as in KO mice compared to the same animals injected with galectin-3 negative Tm1 cells. Gene expression analysis showed that VEGF (vascular endothelial growth factor) mRNA levels were smaller in wild type animals injected with Tm1 galectin-3 positive cells compared to those injected with Tm1 galectin-3 negative cells, indicating that galectin-3 expressed by tumor cells can act as an anti-angiogenic molecule. The present study suggests that galectin-3 can act either as a pro or antitumoral molecule, depending on which type of cell (tumoral or stromal) this lectin is expressed within tumor microenvironment.

INTRODUÇÃO: A propriedade de induzir angiogênese torna o omento um promissor pedículo para pré-fabricar retalhos. OBJETIVO: Estabelecer a área abdominal a ser pré-fabricada por um pedículo omental e analisar o potencial de pré-fabricação (PPF) conforme o tempo de espera entre a introdução do pedículo e a elevação do retalho. MÉTODOS: Foram utilizados 44 coelhos divididos em quatro grupos (A, B, C e D). No grupo A, um fragmento de pele, tecido subcutâneo e músculo cutâneo abdominal foi totalmente separado e ressuturado. Nos demais grupos, um pedículo omental, contendo os vasos gastromentais ligados distalmente, com área equivalente a 9 cm² foi transposto e suturado ao músculo cutâneo abdominal. Um segundo procedimento de incisão e elevação de um retalho contendo pele, subcutâneo e músculo cutâneo abdominal pediculado apenas pelo omento transposto, foi realizado, variando apenas o período de espera entre os dois procedimentos de 7, 21 e 56 dias para os grupos B, C e D respectivamente. Após 15 dias do último procedimento, os retalhos foram visualizados e as áreas viáveis foram calculadas através do programa de computador Image Tool®. Cortes de áreas viáveis foram imunocoradas pelo anti-CD31 para cálculo da densidade microvascular (DMV). RESULTADOS: Os valores médio e máximo das áreas viáveis no grupo D foram respectivamente 45,29 cm² e 99,37 cm² (PPF mediano = 5,03 e PPF máximo = 11,04). Não houve diferença significativa entre as áreas viáveis do grupo D e C. As médias da DMV dos grupos B, C e D foram respectivamente 24,54 vasos/mm², 33,20 vasos/mm² e 27,03 vasos/mm² e maiores do que as médias da DMV das áreas controles de 14,63 vasos/mm², 17,33 vasos/mm² e 18,12 vasos/mm². No grupo A, houve necrose total em todos os retalhos. CONCLUSÃO: O PPF mediano do pedículo omental foi de 5,03 vezes sua área e o tempo de espera para o segundo procedimento foi de, no mínimo, 21dias / INTRODUCTION: The angiogenic induction property of the omentum makes it a promising pedicle to prefabricate flaps. OBJECTIVE: To establish the abdominal area to be prefabricated by the omental pedicle and to analyze the prefabricate potential according to the time delay between the pedicle introduction and the flap release. METHODS: 44 rabbits were divided into four groups (A, B, C and D). In group A, a piece of skin, subcutaneous tissue and abdominal cutaneous muscle has been fully released and sutured again in its place. In other groups, a 9 cm2 omental pedicle containing the gastromental vessels distally tied has been transposed and sutured to abdominal cutaneous muscle. A second procedure, an incision and release of the flap that contained skin, subcutaneous and cutaneous abdominal muscle pediculated only by the omentum, has been carried out. The only variation was the time delay between the two procedures: 7, 21and 56 days for groups B, C and D, respectively. The flaps have been inspected 15 days after the last procedure, and the viable areas have been estimated using the software Image Tool®. The pieces of viable area have been immunostained using anti-CD31 allowing the estimation of the microvascular density. RESULTS: The mean and maximum viable areas in group D were 45.29 cm2 and 99.37 cm2 respectively (average prefabricate potential = 5.03 and maximum prefabricate potential = 11.04). There was no significant difference between the viable areas in groups C and D. The mean microvascular densities of groups B, C and D were 24.54 vessels/mm2, 33.20 vessels/mm2 and 27.03 vessels/mm2 respectively. This was higher than the mean microvascular densities of the control areas, which were 14.63 vessels/mm2, 17.33 vessels/mm2 and 18.12 vessels/mm2. In group A, there were total necrosis in all flaps. CONCLUSION: The prefabricate potential of the omentum was found to be 5.03 times its area and the delay time for the second procedure was, at least, 21 days

INTRODUÇÃO: As adaptações cardiovasculares decorrentes do treinamento físico aeróbio de natação são bem descritas na literatura, entre ela temos a angiogênese. O treinamento físico aeróbio é um dos estímulos que promove angiogênese. Os microRNAs (miRNAs) são uma classe de RNA não codificadora de proteínas de diferentes células em diversos tecidos e estão envolvidos em processos angiogênicos, mas o papel dos miRNAs na angiogênese cardíaca decorrente ao treinamento aeróbico ainda não foi esclarecido. OBJETIVO: Analisar os efeitos de diferentes volumes de treinamento físico de natação sobre a expressão de microRNAs envolvidos na angiogênese cardíaca de ratos. MATERIAIS E MÉTODOS: Ratas Wistar (n=21) foram divididas em grupos Sedentário (SC), Treinado 1 (P1): natação 60min/dia, 5x/sem/10sem, com 5% de sobrecarga, Treinado 2 (P2): mesmo protocolo P1 até a 8ªsem, 9ªsem 2x/dia, e na 10ªsem 3x/dia. Após o período de treinamento, os corações foram retirados e o RNA total foi isolado para a análise da expressão de miRNAs no coração por microarray de miRNA e os miR-126, -let-7f, -221 e -222 foram confirmados por RT-PCR em tempo real. Analisamos ainda os alvos do miR-126, Spred-1 e PI3KR2, e a expressão de proteínas que compõem as vias de sinalização em que esses alvos interferem por Western Blotting. Avaliamos também: Frequência cardíaca (FC) e pressão arterial (PA) por pletismografia caudal, VO2 pico, hipertrofia cárdica (HC) pelo peso do Ventrículo esquerdo/Peso corporal (mg/g), razão capilar/fibra (C/F) por histologia, expressão proteica de VEGF e seus receptores. RESULTADOS: O treinamento aeróbico diminuiu a FC sem alterar a PA, VO2 pico aumentou 11% e 15% em P1 e P2, a HC foi de 17% e 30% em P1 e P2, a razão C/F aumentou 57% e 100% em P1 e P2, acompanhada de um aumento da expressão de VEGF (P1 =42%, P2 =109%). A expressão do miR-let-7f foi aumentada em P2 (140%) comparado aos outros dois grupos (SC = 100%; P1 = 113%), o miR- 221 teve sua expressão diminuida em ambos os grupos treinados comparados xiii ao grupo SC (SC = 100%; P1 = 71%; P2 = 74%), o miR-222 não apresentou diferença na sua expressão entre os grupos (SC = 100%; P1 = 76%; P2 = 81%) e a expressão do miR-126 foi aumentada em P1 (126%) e P2 (142%) comparados ao grupo SC, a expressão de ambos os alvos desse miRNA foi diminuida nos grupos treinados (Spred-1 SC = 100 ± 12,4; P1 = 60 ± 5,6; P2 = 61 ± 8,4; PI3KR2 100 ± 12,3; P1 = 61 ± 12,3; P2 = 21 ± 7,1). Essa diminuição da expressão dos alvos desse miRNA favoreceu um aumento da expressão de proteínas pertencentes as vias de sinalização da PIK3 e MAPKs. CONCLUSÃO: O treinamento aeróbico foi eficaz em promover um aumento da angiogênese cardíaca comprovada por uma maior razão capilar/fibra no coração dos animais treinados e por maior expressão proteica de VEGF, sendo ainda mais evidente nos animais que realizaram um maior volume de treinamento. Os miRNAs relacionados à angiogênese parecem estar envolvidos na regulação desse processo. Além disso, o miR-126 parece ser um dos principais miRNAs envolvidos nesse processo / INTRODUCTION: The cardiovascular adaptations resulting from aerobic physical swimming training are well described in the literature, between these adaptations we have the angiogenesis. The aerobic physical training is one of the stimulus that promotes angiogenesis. The micro RNAs are a class of non coding protein RNAs of different cells in different tissues and are involved in angiogenic processes, but the role of micro RNAs in cardiac angiogenesis due to aerobic training is not clear. OBJECTIVE: To analyze the effects of different volumes of swimming physical training on the expression of micro RNAs involved in angiogenesis in heart of rats. MATERIALS AND METHODS: Wistar female rats (n=21) were divided into groups Sedentary (SC), Trained 1 (P1): 60min/day swimming, 5x/week/10weeks with 5% overload, Trained 2 (P2): same protocol of P1 until the 8th week, 9th week 2x/day, and at 10th week 3x/day. After the training period, the hearts were removed and the total RNA was isolated to analyze the miRNAs expression in the heart by microarray of miRNA and the miRs-126, -let-7f, -221 and -222 were confirmed by real time RT-PCR. We also analyzed the targets miR-126, Spred-1 and PI3KR2, and the protein expression that form the signaling pathways that affect those targets by Western Blotting. We evaluated: heart rate (HR) and blood pressure (BP) by tail plethysmography, peak VO2, cardiac hypertrophy (CH) by weight left ventricle/ corporal weight (mg/g), capillary/fiber (C/P) ratio for histology, VEGF protein expression and its receptors. RESULTS: The aerobic training decreased the HR without change the BP, peak VO2 increased 11% and 15% in P1 and P2, the HR was 17% and 30% in P1 and P2, the ratio C/F increased 57% and 100% in P1 and P2, followed by an increase of VEGF expression (P1=42%, P2=109%). The miR-let-7f had its expression increased in P2 (140%) compared to the other two groups (SC = 100%; P1 = 113%), o miR-221 had its expression decreased in both trained groups compared to SC group (SC = 100%; P1 = 71%; P2 = 74%), the miR-222 showed no difference on its expression between the groups (SC = 100%; P1 = 76%; P2 = 81%) and the miR-126 expression xv was higher in P1 (126%) and P2 (142%) compared to SC group, the expression of both targets of this miRNA was decreased on trained groups (Spred-1 SC = 100 ± 12,4; P1 = 60 ± 5,6; P2 = 61 ± 8,4; PI3KR2 100 ± 12,3; P1 = 61 ± 12,3; P2 = 21 ± 7,1). This decrease of expression of this miRNA targets favor an increase of protein expression belonging to the PIK3 and MAPKs signaling pathways. CONCLUSIONS: The aerobic training was effective on promoting an increased of cardiac angiogenesis comproved by a higher ratio capillary/ fiber on the heart of trained animals and by a higher VEGF protein expression, being even more evident in animals that realized a higher volume training. The miRNAs related to angiogenesis seem to be involved in the regulation of this process. Besides that, the miR-126 seems to be one of the principal miRNA involved on this process

OBJETIVO: demonstrar as alterações histológicas secundárias ao uso de bevacizumabe na aorta descendente de suínos submetida à interrupção dos vasa vasorum. MATERIAIS E MÉTODOS: em doze suínos, divididos em grupos tratamento e controle (n=6 cada) foi realizada dissecção por 5 cm da aorta torácica, ligadura das artérias intercostais e cobertura com polivinil; o grupo tratamento recebeu uma dose endovenosa única de bevacizumabe. Após quinze dias, os animais foram sacrificados para retirada da artéria e preparo das lâminas histológicas dos grupos tratamento, controle e área não manipulada dos dois grupos. As lâminas foram analisadas com relação aos graus de angiogênese, injúria, inflamação e espessamento intimal. A análise estatística foi conduzida através da média e do desvio-padrão dos escores e as comparações entre os grupos foram realizadas pelo teste de Mann-Whitney. Para obtenção de intervalos de confiança de 95% para as médias das contagens dos escores utilizou-se a distribuição de Poisson, a fim de determinar o efeito estatístico. RESULTADOS: o bevacizumabe causou parefeitos em todos os suínos tratados, com um óbito. As variáveis analisadas através da Escala de Magnitude para Efeito Estatístico, demonstram tendência de redução da angiogênese e da injúria e de aumento da inflamação no limite do moderado. Não ocorreu modificação do espessamento intimal entre os grupos. CONCLUSÃO: a medicação utilizada na lesão da parede arterial induzindo hipóxia, mostrou tendência de redução da angiogênese e da injúria, mas não reduziu o processo inflamatório ou o espessamento intimal da parede arterial. O bevacizumabe mostrou toxicidade no modelo suíno. / OBJECTIVE: To demonstrate histological changes secondary to the use of bevacizumab in the descending aorta of pigs after interruption of vasa vasorum flow. METHODS: Twelve pigs were divided into control and treatment groups (n = 6 each). It was performed a 5-cm dissection of the thoracic aorta, ligation of the intercostal arteries and protection with polyvinyl chloride. Pigs in the treatment group received a single intravenous dose of bevacizumab. After 15 days, the animals were euthanized and the aorta removed. Histological slides were prepared for control and treatment groups and for the non-surgically manipulated part of the aorta in both groups. The slides were analyzed for the degree of angiogenesis, injury, inflammation, and intimal thickening. Data were expressed as mean (SD) of scores and groups were compared using the Mann-Whitney test. The Poisson distribution was used to calculate 95% confidence intervals for the mean scores in order to determine effect statistics. RESULTS: Bevacizumab had adverse effects on all treated pigs, leading to one death. The analysis using a scale of magnitudes for effect statistics showed a trend toward a decrease in angiogenesis and injury and an increase in inflammation for moderate effects. There was no change in intimal thickening in either group. CONCLUSION: The medication used for arterial wall injury inducing hypoxia showed a trend toward reduced angiogenesis and injury, but with no reduction in the inflammatory process or intimal thickening of the arterial wall. Bevacizumab showed toxicity in the porcine model.

Chloride intracellular channels (CLICs), 1 and 4 are expressed in endothelial cells where they promote cell proliferation, migration and vessel morphogenesis in vitro. Clic4-/- mice exhibit defects in retinal angiogenesis suggesting CLIC4 functions as an angiogenic regulator. S1P signaling, through S1P receptors S1P1 and S1P2, is essential for endothelial cell functions during vascular development. S1P treatment promotes CLIC4 localization to cell surface suggesting a link between CLICs and S1P pathways. Here we demonstrate that CLICs function in embryonic development, retinal angiogenesis and vascular permeability regulation. Clic1-/-;Clic4-/- embryos die in utero and exhibit severe growth restriction with vascular defects prior to death. Loss of Clic4 in murine endothelium (Clic4ECKO) caused aberrant retinal angiogenesis characterized by reduced vascular outgrowth and increased vessel sprouting. Clic4ECKO mice exhibited increased vessel leakiness as assessed by a lung permeability assay. We establish that CLIC1 and CLIC4 function in distinct branches of the S1P pathway to promote angiogenesis. Knockdown of CLIC1 or CLIC4 in endothelial cells impeded S1P1-mediated induction of AKT and Rac1 and reduced endothelial cell migration and adherence junctions formation. CLIC1 knockdown alone inhibited RhoA activation and actin stress fibers downstream of S1P2. Using pharmacological perturbation of S1P signaling in Clic knockout mice we established that Clic4 is essential for S1P1-mediated regulation of retinal angiogenesis and vascular permeability. We conclude that CLIC1 and CLIC4 function as effectors in the S1P pathway, where they have overlapping functions in S1P1-PI3K signaling and CLIC1 uniquely acts as an effector in S1P2-RhoA signaling cascade. Through these findings, our work defines a molecular mechanism through which CLICs function in endothelium.

Notch signaling plays an important role in developmental and pathological angiogenesis. Notch ligands, Dll4 and Jag1, have been implicated in tumor angiogenesis. Inhibition of Dll4-mediated Notch signaling results in hypersprouting of non-functional vasculature in tumors. We have constructed and analyzed pan-Notch ligand inhibitors, Notch1 decoys 1-24 and 1-36, which are based on the extracellular EGF-like repeats of Notch1. Both Notch1 decoys block angiogenesis in in vitro endothelial cell-based assays and in the mouse retina. We also show that they similarly inhibit Dll4- and Jag1-induced Notch signaling in vitro and result in a significant decrease in tumor growth and tumor vasculature in mouse and human tumor xenograft models. Interestingly, truncated Notch1 decoy variants, Notch1 decoys 1-13 and 10-24, act as ligand-specific Notch inhibitors. Notch1 decoy 1-13 is Dll4-specific whereas Notch1 decoy 10-24 is Jag1-specific. Ligand-specific Notch1 decoys effectively reduce tumor growth in tumor xenograft models in the mouse, including Mm5MT-FGF4, KP1-VEGF, LLC, and B16-F10. Notch1 decoy 1-13 has been demonstrated to increase tumor vasculature by increasing endothelial sprouting and number of tip cells. However, similar to the previously reported effects of Dll4 blockade, the tumor vessels are poorly perfused and hardly functional. On the other hand, Jag1-specific Notch1 decoy 10-24 significantly reduces tumor vessel density and disrupting endothelial-pericyte interactions, causing the impaired vascular structure and attenuated vascular perfusion. In addition, Notch1 decoys 1-13, 10-24, and 1-24 show an anti-metastatic potential in causing a delay of lung metastasis in the B16-F10 tumor model. Unlike gamma-secretase inhibitors and Dll4-blocking agents, Notch1 decoys do not cause GI-associated toxicity or vascular neoplasms. Therefore, our Notch1 decoys may represent a novel alternative and may hold future promise for Notch-targeted cancer therapy.