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Lucitti J.L.,University of North Carolina at Chapel Hill | MacKey J.K.,University of North Carolina at Chapel Hill | Morrison J.C.,University of North Carolina at Chapel Hill | Haigh J.J.,Vascular Cell Biology Unit | And 3 more authors.
Circulation Research | Year: 2012

RATIONALE: The density of native (preexisting) collaterals varies widely and is a significant determinant of variation in severity of stroke, myocardial infarction, and peripheral artery disease. However, little is known about mechanisms responsible for formation of the collateral circulation in healthy tissues. OBJECTIVE: We previously found that variation in vascular endothelial growth factor (VEGF) expression causes differences in collateral density of newborn and adult mice. Herein, we sought to determine mechanisms of collaterogenesis in the embryo and the role of VEGF in this process. METHODS AND RESULTS: Pial collaterals begin forming between embryonic day 13.5 and 14.5 as sprout-like extensions from arterioles of existing cerebral artery trees. Global VEGF-A overexpressing mice (Vegf) formed more, and Vegf formed fewer, collaterals during embryogenesis, in association with differences in vascular patterning. Conditional global reduction of Vegf or Flk1 only during collaterogenesis significantly reduced collateral formation, but now without affecting vascular patterning, and the effects remained in adulthood. Endothelial-specific Vegf reduction had no effect on collaterogenesis. Endothelial-specific reduction of a disintegrin-and-metalloprotease-domain-10 (Adam10) and inhibition of γ-secretase increased collateral formation, consistent with their roles in VEGF-induced Notch1 activation and suppression of prosprouting signals. Endothelial-specific knockdown of Adam17 reduced collateral formation, consistent with its roles in endothelial cell migration and embryonic vascular stabilization, but not in activation of ligand-bound Notch1. These effects also remained in adulthood. CONCLUSIONS: Formation of pial collaterals occurs during a narrow developmental window via a sprouting angiogenesis-like mechanism, requires paracrine VEGF stimulation of fetal liver kinase 1-Notch signaling, and adult collateral number is dependent on embryonic collaterogenesis. © 2012 American Heart Association, Inc. Source


Denecker G.,Inflammation Research Center | Denecker G.,Ghent University | Vandamme N.,Inflammation Research Center | Vandamme N.,Ghent University | And 28 more authors.
Cell Death and Differentiation | Year: 2014

Deregulation of signaling pathways that control differentiation, expansion and migration of neural crest-derived melanoblasts during normal development contributes also to melanoma progression and metastasis. Although several epithelial-to-mesenchymal (EMT) transcription factors, such as zinc finger E-box binding protein 1 (ZEB1) and ZEB2, have been implicated in neural crest cell biology, little is known about their role in melanocyte homeostasis and melanoma. Here we show that mice lacking Zeb2 in the melanocyte lineage exhibit a melanoblast migration defect and, unexpectedly, a severe melanocyte differentiation defect. Loss of Zeb2 in the melanocyte lineage results in a downregulation of the Microphthalmia-associated transcription factor (Mitf) and melanocyte differentiation markers concomitant with an upregulation of Zeb1. We identify a transcriptional signaling network in which the EMT transcription factor ZEB2 regulates MITF levels to control melanocyte differentiation. Moreover, our data are also relevant for human melanomagenesis as loss of ZEB2 expression is associated with reduced patient survival. © 2014 Macmillan Publishers Limited All rights reserved. Source


Gembarska A.,Center for the Biology of Disease | Gembarska A.,Catholic University of Leuven | Luciani F.,Center for the Biology of Disease | Luciani F.,Catholic University of Leuven | And 29 more authors.
Nature Medicine | Year: 2012

The inactivation of the p53 tumor suppressor pathway, which often occurs through mutations in TP53 (encoding tumor protein 53) is a common step in human cancer. However, in melanoma-A highly chemotherapy-resistant disease-TP53 mutations are rare, raising the possibility that this cancer uses alternative ways to overcome p53-mediated tumor suppression. Here we show that Mdm4 p53 binding protein homolog (MDM4), a negative regulator of p53, is upregulated in a substantial proportion (∼65%) of stage I-IV human melanomas and that melanocyte-specific Mdm4 overexpression enhanced tumorigenesis in a mouse model of melanoma induced by the oncogene Nras. MDM4 promotes the survival of human metastatic melanoma by antagonizing p53 proapoptotic function. Notably, inhibition of the MDM4-p53 interaction restored p53 function in melanoma cells, resulting in increased sensitivity to cytotoxic chemotherapy and to inhibitors of the BRAF (V600E) oncogene. Our results identify MDM4 as a key determinant of impaired p53 function in human melanoma and designate MDM4 as a promising target for antimelanoma combination therapy. © 2012 Nature America, Inc. All rights reserved. Source


Nguyen A.H.T.,McGill University | Tremblay M.,McGill University | Haigh K.,Ghent University | Haigh K.,Vascular Cell Biology Unit | And 8 more authors.
Human Molecular Genetics | Year: 2013

Loss of the tumor suppressor PTEN is a common occurrence in prostate cancer. This aberration leads to the ectopic activation of the PI3K-Akt pathway, which promotes tumor growth. Here, we show that the transcription factor Gata3 is progressively lost in Pten-deficient mouse prostate tumors as a result of both transcriptional down-regulation and increased proteasomal degradation. To determine the significance of this loss, we used conditional loss- and gain-of-function approaches to manipulate Gata3 expression levels in prostate tumors. Our results show that Gata3 inactivation in Pten-deficient prostates accelerates tumor invasion. Conversely, enforced expression of GATA3 in Pten-deficient tissues markedly delays tumor progression. In Pten-deficient prostatic ducts, enforced GATA3 prevented Akt activation, which correlated with the down-regulation of Pik3cg and Pik3c2a mRNAs, encoding respectively class I and II PI3K subunits. Remarkably, the majority of human prostate tumors similarly show loss of active GATA3 as they progress to the aggressive castrate-resistant stage. In addition, GATA3 expression levels in hormone-sensitive tumors holds predictive value for tumor recurrence. Together, these data establish Gata3 as an important regulator of prostate cancer progression.© 2013. Published by Oxford University Press. All rights reserved. Source


Maes C.,Catholic University of Leuven | Araldi E.,Harvard University | Haigh K.,Vascular Cell Biology Unit | Haigh K.,Ghent University | And 7 more authors.
Journal of Bone and Mineral Research | Year: 2012

Fetal growth plate cartilage is nonvascularized, and chondrocytes largely develop in hypoxic conditions. We previously found that mice lacking the hypoxia-inducible transcription factor HIF-1α in cartilage show massive death of centrally located, hypoxic chondrocytes. A similar phenotype was observed in mice with genetic ablation of either all or specifically the diffusible isoforms of vascular endothelial growth factor (VEGF), a prime angiogenic target of HIF-1α. Here, we assessed whether VEGF is a critical downstream component of the HIF-1α-dependent survival pathway in chondrocytes. We used a genetic approach to conditionally overexpress VEGF164 in chondrocytes lacking HIF-1α, evaluating potential rescuing effects. The effectiveness of the strategy was validated by showing that transgenic expression of VEGF164 in Col2-Cre;VEGF f/ f mice stimulated angiogenesis in the perichondrium, fully corrected the excessive hypoxia of VEGF-deficient chondrocytes, and completely prevented chondrocyte death. Yet, similarly crossed double-mutant embryos lacking HIF-1α and overexpressing VEGF164 in the growth plate cartilage still displayed a central cell death phenotype, albeit slightly delayed and less severe compared with mice exclusively lacking HIF-1α. Transgenic VEGF164 induced massive angiogenesis in the perichondrium, yet this only partially relieved the aberrant hypoxia present in HIF-1α-deficient cartilage and thereby likely inflicted only a partial rescue effect. In fact, excessive hypoxia and failure to upregulate phosphoglycerate-kinase 1 (PGK1), a key enzyme of anaerobic glycolytic metabolism, were among the earliest manifestations of HIF-1α deficiency in cartilaginous bone templates, and reduced PGK1 expression was irrespective of transgenic VEGF164. These findings suggest that HIF-1α activates VEGF-independent cell-autonomous mechanisms to sustain oxygen levels in the challenged avascular cartilage by reducing oxygen consumption. Hence, regulation of the metabolic pathways by HIF-1α and VEGF-dependent regulation of angiogenesis coordinately act to maintain physiological cartilage oxygenation. We conclude that VEGF and HIF-1α are critical preservers of chondrocyte survival by ensuring an adequate balance between availability and handling of oxygen in developing growth cartilage. Copyright © 2012 American Society for Bone and Mineral Research. Source

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