Mechanisms involved in quiescent blood vessel homeostasis

Abstract

Our vascular system is an organized and hierarchical blood vessel network lined by a monolayer of endothelial cells (ECs) that supplies oxygen and nutrients to all tissues and organs in our body. Importantly, they are not passive conduits for blood flow and they contribute to organ physiology and homeostasis throughout the entire life of the organisms. Therefore, it is not surprising that an imbalance of this vascular network is involved the pathogenesis of many diseases such as cancer, stroke or myocardial infarction. The Notch signalling pathway is a critical regulator in the process of angiogenesis, participating in the tip-stalk specification, arterial-venous differentiation, vessel stabilization and maturation. Importantly, pharmacological compounds targeting distinct members of the Notch signalling pathway have been used in the clinics but their effect on the homeostasis of different types of blood vessels is unknown. In this thesis, we have developed and used a wide range of novel genetic tools, loss-offunction mouse models, imaging, trancriptomic and proteomic approaches to uncover the role of several Notch signalling members in the regulation of organ-specific vascular homeostasis at high cellular and molecular resolution. We found that Dll4/Notch signalling is active in most quiescent endothelium of several organs, such as the heart, lung, liver and brain. However, despite being active in all the mentioned vascular tissues, only in heart and liver vessels it plays an essential role in the maintenance of vascular quiescence by actively repressing EC proliferation. By using comparative transcriptomic and proteomic approaches, we found that Dll4/Notch1 actively supresses the Myc pathway to sustain vascular quiescence in the liver. By analysing a series of Notch signalling mutants, we found that while loss of the ligand Dll4 leads to an intermediate increase in p-ERK levels and cell cycle entry, resulting in EC hyperproliferation; loss of the co-factor Rbpj or the receptor Notch1 induces even higher ERK signalling activity but not EC proliferation. At high p-ERK levels there is an increase in the expression of the cell cycle inhibitor p21, which arrests EC proliferation. These arrested cells have also several features of endothelial senescence. In addition, we observed that the loss of Dll4/Notch signalling in liver ECs, promotes the transient increase in the proliferation of neighboring hepatocytes prior to their own expansion, suggesting the existence of angiocrine factors secreted by ECs after the loss of Notch signalling. Moreover, the effect of Notch on liver endothelial cell proliferation was very heterogenous and zonated. ECs located close to the central veins had a higher proliferation potential compared to the endothelium located in oxygen-rich periportal zones. Using multispectral genetic mosaics, we also identified a rare population of liver ECs that is able to clonally expand more than most of their neighbours, suggesting the existence of liver-resident endothelial progenitor cells. In addition to these studies on the role of Notch in vascular biology, we also used and validated an inducible dual reporter-Cre mouse allele (iSuRe-Cre), which by significantly increasing Cre activity in reporter-expressing cells, it provides certainty that these cells have completely recombined floxed alleles.