Molecular dissection of the role of ARAP3 in angiogenesis

Lead Research Organisation: Babraham Institute
Department Name: Dept of Development & Signalling

Abstract

During embryonic development, specialised tissues form. For example, a circulatory system is required to nourish the developing organism. This process is called angiogenesis. Angiogenesis is also important in cancer, where new tumours need to be provided with blood so they can be nourished. We found out that ARAP3, a new signalling protein, is needed for angiogenesis when we studied a mouse that lacked ARAP3 and saw that the embryos died because they couldn’t form blood vessels. We want to establish why ARAP3 is required for angiogenesis, and whether it could be a good target for cancer treatment. For this, we will look at the way in which ARAP3 is controlled and in term controls other cellular proteins. We will analyse vessels in mouse embryos, which either lack ARAP3 or where it contains a small change (mutation). Secondly, we will study endothelial cells (which form blood vessels) lacking ARAP3, or containing the mutant ARAP3 protein, to see how they differ from normal cells. Finally, we will put ARAP3, or its mutants back into endothelial cells, to ‘rescue’ them. In this way we will learn why ARAP3 is required for angiogenesis, and whether it is a good cancer drug target.

Technical Summary

Phosphoinositide 3-OH kinases (PI3Ks) control a multitude of fundamental cellular responses after appropriate stimulation. One such response is angiogenesis, both during embryogenesis and in tumorigenesis. ARAP3 was identified in a screen for novel PI3K effectors as a PI3K- and Rap- regulated dual GTPase activating protein (GAP) for Rho and Arf. To understand its physiological function, we recently created an ARAP3 knock-out mouse. Loss of ARAP3 leads to embryonic lethality in mid-gestation: ARAP3-/- embryos are retarded, anaemic and devoid of blood vessels; their yolk sacs contain no blood vessels and their placentas do not develop the labyrinth of maternal and foetal capillaries. We conclude that embryonic death is due to an angiogenesis defect, hence ARAP3 is required for angiogenesis in the developing embryo. To elucidate the signalling upstream of ARAP3 in the control of angiogenesis, we created a second mouse model (ARAP3R302/3A), in which ARAP3 can no longer be activated by PI3K. We propose to analyse embryogenesis in ARAP3R302/3A embryos and compare them to ARAP3-/- embryos. We propose further to analyse endothelial cells derived from ARAP3-/- and ARAP3R302/3A embryos in terms of their shape, cellular junctions, focal adhesions, ability to chemotax and to form tubular structures in vitro, to define the precise cellular defects arising from lack (or mutation) of ARAP3. Together, these studies will establish the importance of ARAP3 as an effector of PI3K for control of angiogenesis. To clarify the signalling pathways downstream of ARAP3, we will dissect out whether particular defects are due to ARAP3?s Rho or Arf GAP activity. This will be done by rescuing ARAP3-/- endothelial cells by retroviral transduction with individual ARAP3 GAP domain point mutants. Thereby, we will increase our understanding of the cross-talk between Rho and Arf small GTPases, which is mediated by ARAP3 and controlled by PI3K during angiogenesis. Furthermore, we will address modulation of ARAP3?s Rho GAP activity by Rap by rescue with a Ras binding domain point mutant; to see whether ARAP3?s scaffold function is important for its angiogenic potential, we will use a ?SAM domain truncation construct. In summary, this project will elucidate PI3K-controlled signalling to Rho and Arf GTPases mediated by ARAP3 in angiogenesis in developing embryos and in endothelial cells. Whilst our work concentrates on embryogenesis, the results obtained are likely to be applicable to the angiogenic processes fundamental to the growth of a range of human cancers.
 
Description Our findings were presented to scientific audiences at conferences and by publication in peer reviewed journals. We presented to the wider public by press releases, that were picked up by some local publications, and I gave a podcast interview with Science Signaling, that can still be accessed online. This was basic biomedical research, and whilst the work will be indirectly contributing to the types of impacts that are listed below, immediate, direct impacts cannot easily be attributed
First Year Of Impact 2010
Sector Pharmaceuticals and Medical Biotechnology
 
Description Project Grant
Amount £457,820 (GBP)
Funding ID MR/M023060/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 09/2015 
End 08/2018
 
Description Wellcome Trust University of Edinburgh Institutional Support Fund
Amount £34,800 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Institutional Strategic Support Fund
Sector Charity/Non Profit
Country United Kingdom
Start 04/2014 
End 10/2015
 
Title Angiotool 
Description AngioTool is a software tool for quantitative analysis of microscopic images depicting complex vascular networks. To our knowledge there was no such tool available before. We published an open access article describing AngioTool to increase awareness in the scientific community (see papers) 
Type Of Material Data analysis technique 
Year Produced 2011 
Provided To Others? Yes  
Impact This software tool was made freely available to the public and was published as an open access research paper as well. It appears to be widely used in the research community and has been cited in numerous other research papers that use it for analysis of their vascular networks. 
URL https://ccrod.cancer.gov/confluence/display/ROB2/Home
 
Description International collaboration with NIH National Cancer Institute 
Organisation National Institutes of Health (NIH)
Department National Cancer Institute (NCI)
Country United States 
Sector Public 
PI Contribution The research pursued for this fellowship has resulted in an international collaboration which already led to publications in peer-reviewed journals.
Collaborator Contribution intellectual input
Impact two research papers and a book chapter
Start Year 2009
 
Description allantois explants 
Organisation National Institutes of Health (NIH)
Department National Cancer Institute (NCI)
Country United States 
Sector Public 
PI Contribution We provided micrographs of allantois explants for generation and optimisation of this new tool
Collaborator Contribution Generation of a computational analysis tool for complexity of networks generated by sprouting angiogenesis
Impact Pub Med ID: 20978237 This collaboration involves biologists and a mathematician
Start Year 2009
 
Description Press Release Science Signalling 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Our recent Science Signaling paper was chosen for broadcasting by podcast by the editors of Science Signaling. After recording the interview, we also put together a press release, to co-incide with the paper appearing. We had further enquiries from several journalists. I understand the work was cited in some local papers and in 'Business Weekly'.

This has publicised our work more widely than the publication in a scientific journal alone would have.
Year(s) Of Engagement Activity 2010
URL http://stke.sciencemag.org/content/suppl/2010/10/22/3.145.pc19.DC1
 
Description Schools Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 10 GSCE / 'A'-level student came to the lab, listened to a short talk and participated in a hands-on practical experience.

Some of the students provided positive feed-back on the visit to the lab and said it had helped them with their career choice
Year(s) Of Engagement Activity 2008,2009,2010,2011