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Epistasis analysis of angiogenes with high cellular definition

Periodic Reporting for period 4 - AngioGenesHD (Epistasis analysis of angiogenes with high cellular definition)

Reporting period: 2019-09-01 to 2020-02-29

Blood vessels have an important role in cancer and cardiovascular diseases. It is well established that inhibition of vascular growth or angiogenesis, decreases the growth of tumors by reducing its blood and oxygen supply. In cardiovascular ischemic disease, such as after myocardial infarction, there is also interest in promoting vascular function, arterialization and growth, in order to improve heart function. Cancer and cardiovascular diseases are the most common causes of morbidity and death around the world and blood vessels play a very important role in their progression. Therefore, understanding how to therapeutically manipulate the biology and function of blood vessels is of great biomedical relevance.
In our ERC StG AngioGenesHD project we proposed to use novel genetic and imaging technologies in order to understand how the Notch and VEGF signaling pathways regulate different processes related with endothelial differentiation and proliferation during vascular development, homeostasis and associated diseases. The Notch and VEGF signalling pathways are key regulators of vascular biology and the deeper understanding of their context-dependent functions can enable a more effective blockade or induction of angiogenesis and surrounding tissue growth.
Overall the project was very successful. We developed genetic technology of broad utility, patented a part of it, established numerous collaborations, and identified molecular and cellular mechanisms of high biological and therapeutic relevance. This is evident by the group publlication record in the last 5 years and the participation in numerous international scientific conferences and science dissemination events.
One of the most important aspects of our ERC StG AngiogenesHD project was technology development. We have generated new mouse models of broad applicability to study combinatorial gene function in a mosaic fashion and with much higher cellular and temporal resolution (Fig. 1), during organ development or in disease (Pontes-Quero et al., 2017 Cell). In classical genetics, only one gene function is analysed at a time. With our new models and methods, up to 6 different gene functions can be analysed in an indvidual or combinatorial manner, and all with single-cell or clonal resolution. We also developed a new mouse model that greatly increase the ease, efficiency, and reliability of Cre-dependent conditional mutagenesis and gene function analysis (Fernandez-Chacon et al., 2019 NCOMM). This is the most widely used method to manipulate and understand gene function in biomedical research.
Using these novel advanced mouse models generated in our laboratory, we could also indentify biological mechanisms of high relevance. One of the first discoveries concerned the counterintuitive finding that high mitogenic stimulation arrests angiogenesis. The existent view was that an increase in growth factor concentration, and the resulting mitogenic activity, increases both endothelial proliferation and sprouting. Our results instead indicated that high mitogenic stimulation induced by VEGF, or Notch inhibition, arrests the proliferation of angiogenic vessels (Fig. 2). This is due to the existence of a bell-shaped dose-response to VEGF and MAPK activity that is counteracted by Notch and p21, determining whether endothelial cells sprout, proliferate, or become quiescent. The identified mechanism highlights that more angiogenic growth factors may not result in the intended result and arrest vascular proliferation. This should be considered to achieve optimal therapeutic modulation of angiogenesis. We are now using the molecular and mechanistic knowledge obtained to induce angiogenesis more effectively. We believe that by targeting some of the identified hypermitogenic arrest mechanisms we can boost vascular growth.
On another related project, we also found that the development of arteries can occur without the need for transcription factors previously thought to be involved in the direct differentiation of arteries. We found that these factors are not needed if endothelial cell growth and metabolism is supressed. Our findings indicate that arterialization involves the timely supression of cell growth/metabolism and not a change in cell identity, fate or genetic program (Fig. 3). This changes our view of the arterialization process and may enable a better induction of this process during tissue growth, regeneration or in ischemic cardiovascular disease. From a translational standpoint, is known that atherosclerosis is an artery-specific disease, with no incidence in veins, and that coronary artery bypass surgery is more likely to fail when the graft is a heterologous saphenous vein rather than an artery. Induction of collateral arterial growth is also seen as a promising new approach against cardiac ischemia caused by coronary artery disease. In this context, the ability to induce or reprogram the arterial or venous identities of established and quiescent vessels is of great interest and our project provides important mechanistic insights that can be used to achieve this.
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