Vascular Development

Research Interests

The group research pursues a range of studies related to the signaling mechanisms regulating vascular development. We aim to better understand the molecular bases underlying cell-cell communication, cell fate decision, adhesion and migration during normal blood vessel morphogenesis/angiogenesis and to gain insight into their post-natal roles and their relevance for cancer and other disease conditions associated with neovascularization.

Group Members

Research Project

Arteriogenesis: identification of new members of the Notch pathway involved in arterial cell fate determination

Notch receptors and their ligands, Delta-like (Dll) and Jagged (Jag), participate in an evolutionary conserved signaling pathway that functions to modulate cell-fate decisions of a variety of cell types originating from all three germlayers. Mutations in humans, mice and zebrafish demonstrated the importance of Notch signaling in the regulation of vascular development.
In zebrafish, Notch signaling is required for arterial identity by suppressing the venous fate in developing artery cells. In mice, Notch4 and Dll4 are specifically expressed in arterial endothelial cells, suggesting a similar role. Knockout of the Notch4 receptor gave no obvious phenotype alone, although Notch1/Notch4 double mutant embryos show severe vascular remodeling defects, also observed in Hey1/Hey2 double mutants. In contrast, we have shown that the Dll4 ligand ALONE is required in a dosage-sensitive manner for normal arterial patterning in development. We observed lethal haploinsufficiency in Dll4+/- embryos, with disrupted vascular network and aortic atrophy. Dll4 overexpression, on the other hand, caused aortic hypertrophy and loss of vascular identity. In the Dll4 knockout embryos we observed ectopic expression of venous markers in the dorsal aortae whereas in the Dll4 overexpression transgenics arterial markers were expressed in the cardinal veins.
These two complementary phenotypes are suggestive of an important role for Dll4 in the development of the vascular bed, in particular in arteriogenesis, where it may be responsible for the activation of genes involved in the establishment of the arterial endothelial cell phenotype. Although there is compelling evidence for such a role, almost nothing is yet known on its mechanistic basis. It is this void which we will attempt to fill by discovering the downstream genes that are responsible for mediating the effect of Notch activation in the endothelial cells.
We propose to characterize the gene expression profile of endothelial cells in these two mutant strains by microarray analysis and carry out expression pattern characterization in order to identify novel genes situated downstream of Notch signaling in mammalian arteriogenesis. We hope that our understanding of the molecular mechanisms by which Notch regulates arterial/venous specification may provide insights into the pathological angiogenesis that support cancer growth and novel tools potentially useful to induce arteriogenesis in ischemic tissues.

Funding

Fundação para a Ciência e a Tecnologia (FCT) Project Grant, Portugal

Research Project

Notch signaling function in physiological and tumour angiogenesis

The great sensitivity of the embryonic vasculature to the Notch ligand Delta-like 4 (Dll4) levels raises the possibility that it may constitute a good target for therapeutic intervention in adult tumour-induced neoangiogenesis. Furthermore, its expression is greatly increased in the vasculature of xenografted as well as endogenous human tumours and was shown to be induced by hypoxia, a condition frequently present in the tumour milieu. Nevertheless, the mechanistic basis of Dll4 function in general, and in the tumour angiogenesis context in particular, remains to be better studied.
The embryonic lethal phenotype of Dll4 null mutants makes it impossible to characterize its postnatal function in vivo using conventional gene targeting. In this project we therefore propose to use inducible gene deletion and gene overexpression strategies to create loss- and gain-of-function mutants to address the various aspects of Dll4 function in vivo in newborn and adult mice. The genetically modified mouse lines already established in our laboratory enable us to carry out a number of in vivo experiments that will help dissect the role of Dll4/Notch signaling in the regulation of vascular branching, specification of tip cell identity, as well as bone marrow-derived endothelial progenitor cell differentiation under both physiological and tumour conditions.

Funding

Fundação para a Ciência e a Tecnologia (FCT) Project Grant, Portugal

Publications

(Selected) Update March (2009).

Benedito, R., Trindade, A., Hirashima, M., Henrique, D., Lopes-da-Costa, L., Rossant, J., Gill, P.S., Duarte, A. (2008). Loss of Notch signalling induced by Delta-like 4 (Dll4) causes arterial calibre reduction by increasing endothelial cell response to angiogenic stimuli BMC Dev Biol 8(1) :117-125

Koch, U., Fiorini, E., Benedito, R., Besseyrias, V., Gossler, K., Pierres, M., Manley, N.R., Duarte, A., MacDonald, R., Radtke, F. (2008). Delta-like 4 is the essential, non-redundant ligand for Notch 1 during thymic T cell lineage commitment J Exp Med 205(11) :2515-23

Tammela, T., Waltari, M., Wirzenius, M., Suchting, S., Schomber, T., Murtomäki, A., Hellström, M., Haiko, P., Duarte, A., Ylä-Herttuala, S.Gerhard Christofori, G., Laakkonen , P., Betsholtz, C., Eichmann, A. and Alitalo, K. (2008). Inhibition of VEGFR-3 signaling suppresses angiogenic sprouting and vascular network formation Nature 454(7204) :656-60

Suchting S, Freitas C, le Noble F, Benedito R, Bréant C, Duarte, A, Eichmann A. (2007). Negative regulators of vessel patterning Novartis Found Symp 283 :77-80

Schenet, J.S., Jiang, W., Kumar, S.R., Krasnoperov, V., Trindade, A., Benedito, R., Djokovic, D., Borges, C., Ley, E.J, Duarte, A. and Gill, P.S. (2007). Inhibition of Dll4 mediated signaling induces proliferation of immature vessels and results in poor tissue perfusion Blood 109(11) :4753-60

Suchting, S., Freitas, C., le Noble, F., Benedito, R., Breant, C., Duarte, A., Eichmann, A. (2007). The Notch ligand Delta-like 4 negatively regulates endothelial tip cell formation and vessel branching Proceedings of the National Academy of Science USA 104(9) :3225-30

Seo S, Fujita H, Nakano A, Kang M, Duarte A, Kume T. (2006). The forkhead transcription factors, Foxc1 and Foxc2, are required for arterial specification and lymphatic sprouting during vascular development Dev Biol 294(2) :458-70

Benedito, R. & Duarte, A. (2005). Expression of Dll4 during mouse development suggests multiple developmental roles Gene Expression Patterns 5 :750-5

Duarte, A, Hirashima, M., Benedito, R., Trindade, A., Diniz, P., Lopes da Costa, L., Henrique, D. & Rossant, J. (2004). Dosage sensitive requirement for mouse Dll4 in artery development Genes & Development 18 :2472 - 2478