Molecular basis of artery vein differentiation

Understanding the mechanisms that control growth and differentiation of blood vessels has important clinical implications. For example, abnormal vascular growth in the retina is a major complication in diseases such as diabetic retinopathy or age related macular degeneration and is the leading cause of blindness in the Western world. However, therapeutic intervention can only be achieved if we fully understand the biological principles of vascular development. Progress has been made in the last decade and some of the basic stop-and-go signals of vessel growth have been identified, but the mechanisms that lead to the complexity and diversity of a fully differentiated vascular tree are not well understood.

In this project we investigate how vessels differentiate into arteries or veins. To this end we use mice that have conditional mutations in certain genes, suspected to play a role in this process. The mutations can be activated with a drug (tamoxifen) allowing us to study the function of these genes at specific time points during development. This will help us to understand why the two vessel types behave differently during development and under pathological conditions. Such knowledge will form the basis for future therapies that target only specific blood vessel types and might allow us to manipulate the shape of the vascular tree.

The vasculature is a highly organized and complex structure on which each and every cell in the body depends. During development, primitive blood vessels form initially simple plexuses that subsequently remodel and differentiate into mature networks consisting of arteries, veins and capillaries. Understanding the molecular mechanisms that guide this process is highly relevant for therapeutic angiogenesis as well as for antiangiogenic therapy. Recent studies in zebrafish have shown that signaling via Notch and vascular endothelial growth factor (VEGF) plays a role in dorsal aorta and cardinal vein formation (the first vessels to develop), but the factors that control artery vein (AV) differentiation later in development are less well understood. A number of genes, suspected to play a role in AV differentiation, have been knocked out in mice but these experiments suffer from the problem that mutations affecting the vasculature usually lead to early embryonic death precluding the study of vascular differentiation in older animals. Here we address this problem by applying an inducible gene deletion method. We recently created mice that express tamoxifen-inducible Cre recombinase specifically in endothelial cells. When they are crossed with mice that contain loxP sites in a particular gene, tamoxifen injection will delete this gene. Preliminary tests have shown that we can target close to 100% of endothelial cells in postnatal animals. We intend to use this system to delete four different genes, involved in Notch and VEGF signaling. We will address the role of VEGF signaling in AV differentiation by deleting VEGF receptor 2 (VEGFR2) and the artery specific VEGF co-receptor Neuropilin-1. We will also study notch signaling (believed to be downstream of VEGF in AV differentiation) by deleting the artery specific notch ligand Delta-like-4 (Dll4). Furthermore, we have previously found evidence that oxygen levels can influence Dll4 expression in arteries and we will therefore target oxygen sensing in arteries by deleting the von-Hippel-Lindau gene. In summary we aim to establish the role of these four genes in postnatal artery vein differentiation but also possible genetic interactions between them.