Arterialization is a process that describes the development of arteries from blood capillaries. Needless to say, stimulating this process in the ischemic heart to restore blood flow and limit damage is a holy grail of translational biomedicine. Designing approaches to promote arterialization requires a detailed knowledge of the pathways involved in the process. Probably most is known about the role played by the vascular endothelial growth factor (VEGF) and Notch signalling pathways, as an intricate balance between these two pathways has been shown to determine the formation of the vascular plexus (primitive vasculature) in the retina. Thereafter, primitive vessels take on the characteristics of arteries and veins and capillaries that are surplus to requirement and not efficiently perfused, regress and disappear. Notch signalling, which involves the release and nuclear translocation of the Notch intracellular domain (NICD) to interact with the transcription factor RBPJ, plays a key role in this process, and without it, arterial specification and development fail.1,2 Thus, Notch signalling has been assumed to dictate the genetic changes required to reprogram a sub-set of capillary endothelial cells to differentiate, migrate, and eventually form arteries.
The study by Luo et al.3 makes a major contribution to our understanding of the process of arterialization. It is, however, not an easy read and our journal club took 3 weeks to get to grips with the complex loss- and gain-of-function genetic mosaic mouse models that helped the authors determine the consequences of specific competition between cells with high versus low Notch signalling. The story begins with a prior publication from the same group in which they demonstrated that contrary to popularly held opinion, high mitogenic stimulation induced by VEGF, or Notch inhibition, actually halts the proliferation of angiogenic vessels.4 This led the authors to hypothesize that alterations in the cell cycle rather than genetic differentiation contribute to the process of arterialization. Using a well thought out set of endothelial cell mosaic mice with normal low and high Notch signalling activity (all in different colours) the authors studied the development of the cardiac vasculature. Arterialization was not affected by this manipulation which allowed the authors to study the fates of the cells with distinct differences in Notch activity. This led, for example to realization that endothelial cells with high Notch signalling were outcompeted during the development of coronary vessels but not during the development of the endocardium. VEGFR2 mosaic mice were also used to investigate the influence of VEGF and extracellular regulated kinase signalling on arterialization. This revealed that cell with low VEGFR2 signalling did not form arteries; however, but did form the endocardium and heart-surface venous vessels. Endothelial cells with increased VEGFR2 signalling, on the other hand, frequently formed arteries in preference to veins. There are too many mice to go into all the details here but the authors take home message is that Notch function is important only in the capillary-to-arterial transition, a process associated with a timely reduction in metabolic and cell-cycle activity. Also, Notch does not directly induce arterial differentiation, but instead reduces Myc-dependent metabolic and cell-cycle activity. In fact, it seems to be the timely inhibition of Myc and the cell cycle that is essential for arterialization.
The paper by Luo et al.3 is a milestone for our understanding of the events that link proliferation and differentiation during arterialization. There are also clear implications for translation as the realization of the temporal activation and inactivation of VEGF, Notch and Myc signalling may help design approaches to activate arterialization in ischemic cardiovascular diseases.
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