Regulatory pathways controlling venous identity
Lead Research Organisation:
University of Oxford
Department Name: Ludwig Institute for Cancer Research
Abstract
The circulatory system of mammals is dependent on the formation of two distinct types of blood vessel, arteries and veins. The differences between these two blood vessel types arise due to alternative molecular signals early on in development. However, very little is known about these signals and how vein-specific genes are switched on in veins and off in arteries. Understanding this process is crucial for understanding and treating cardiovascular disease. For example, veins are often used to replace damaged arteries in coronary bypass surgery but often do not correctly adapt, and defective arterial-venous differentiation can lead to enlarged blood vessels at risk of catastrophic ruptures.
This study will look at regions of DNA called enhancers. These enhancer regions control whether a gene is on or off through the binding of proteins called transcription factors. We have found two enhancers which can switch genes on specifically in veins. This project aims to find what transcription factors bind to, and control, these vein-specific enhancers, and to understand what external signals cause this to happen. We will also use these gene switches to make an animal model capable of deleting any gene of interest specifically from veins.
This study will look at regions of DNA called enhancers. These enhancer regions control whether a gene is on or off through the binding of proteins called transcription factors. We have found two enhancers which can switch genes on specifically in veins. This project aims to find what transcription factors bind to, and control, these vein-specific enhancers, and to understand what external signals cause this to happen. We will also use these gene switches to make an animal model capable of deleting any gene of interest specifically from veins.
Technical Summary
This project will use the identification, analysis and characterisation of two novel venous-specific gene enhancers and their cognate binding transcription factors to understand the transcriptional and signalling pathways that regulate venous identity. While research clearly demonstrates that venous identity in endothelial cells is actively acquired, very little is known about the pathways that control this process. Ephb4 and COUP-TFII play essential roles in the specification and differentiation of veins, yet the manner in which their expression is regulated in venous endothelial cells is not known. In fact, it is still unclear whether their transcription is actively stimulated in veins, or repressed in arteries. Interpreting the results of signalling experiments is still challenging, due to a lack of even basic knowledge of venous gene regulation. The project proposed here will expand our knowledge of venous gene regulation by identifying and analysing enhancers driving Ephb4 and Coup-TFII expression in venous endothelial cells, delineate the key cis-motifs and trans-factors that regulate these enhancers in an unbiased manner, and discern the vascular signalling pathways upstream of this regulation.
Planned Impact
Virtually nothing is known about how venous endothelial cells become specified and differentiation. Consequently, this research is anticipated to have a major influence in the vascular developmental biology field and far beyond. This work will be of interest to a wide range of academics, described in detail in the Academic Beneficiary section. In addition, this research will provide key insights into how venous identity is both acquired and maintained, which is anticipated to open new avenues of research into mechanisms that may alter blood vessel identity for therapeutical benefits. For example, this work will be of interest to teams looking to manipulate cells to take on an endothelial, venous or arterial fate with the aim of assisting in repair after ischemic damage.
In addition, although the proposed project is in basic research, understanding how blood vessels take on, and potential can loose, venous identify has potential interest to health care professionals. For example, veins are often used to repair damaged arteries in the heart, and this research may illuminate mechanisms beneficial or detrimental to the survival, and adaptability, of these grafts. The identification of key factors directing venous fate may also provide novel biomarkers to assess the differentiation status of vessels after vascular disease or surgery, and to identify cases of venous graft failure.
In addition, although the proposed project is in basic research, understanding how blood vessels take on, and potential can loose, venous identify has potential interest to health care professionals. For example, veins are often used to repair damaged arteries in the heart, and this research may illuminate mechanisms beneficial or detrimental to the survival, and adaptability, of these grafts. The identification of key factors directing venous fate may also provide novel biomarkers to assess the differentiation status of vessels after vascular disease or surgery, and to identify cases of venous graft failure.
Publications
Becker PW
(2016)
An Intronic Flk1 Enhancer Directs Arterial-Specific Expression via RBPJ-Mediated Venous Repression.
in Arteriosclerosis, thrombosis, and vascular biology
Chiang IK
(2017)
SoxF factors induce Notch1 expression via direct transcriptional regulation during early arterial development.
in Development (Cambridge, England)
Chiang IK
(2017)
Correction: SoxF factors induce Notch1 expression via direct transcriptional regulation during early arterial development. Development doi: 10.1242/dev.146241.
in Development (Cambridge, England)
Lupu IE
(2020)
Coronary vessel formation in development and disease: mechanisms and insights for therapy.
in Nature reviews. Cardiology
Neal A
(2019)
Venous identity requires BMP signalling through ALK3.
in Nature communications
Neal A
(2021)
ETS factors are required but not sufficient for specific patterns of enhancer activity in different endothelial subtypes.
in Developmental biology
Payne S
(2023)
Transcription factors regulating vasculogenesis and angiogenesis.
in Developmental dynamics : an official publication of the American Association of Anatomists
Payne S
(2018)
Endothelial-Specific Cre Mouse Models.
in Arteriosclerosis, thrombosis, and vascular biology
Payne S
(2019)
Regulatory pathways governing murine coronary vessel formation are dysregulated in the injured adult heart.
in Nature communications
Description | Blood vessels help supply the body with fuel and to remove waste. Endothelial cells line the inside of every blood vessel. We were interested in how the endothelial cells in veins (which carry the blood back to the heart) become different than arterial endothelial cells. This is important to know, as the loss of endothelial identity is associated with my different diseases. Further, we often wish to manipulate blood vessel growth (e.g. to get rid of blood vessels supplying tumours with energy, or to grow new vessels in damaged tissues to help regeneration) and this is impossible when we do not understand them properly. Our work identified and characterised two vein-specific enhancers for the Ephb4 and Coup-TFII genes. Analysis of these enhancers has uncovered the entirely unsuspected regulatory pathway which controls venous endothelial cell identity: the BMP4 ligand signals through the BMPR1a receptor to the SMAD1/5 transcription factors, which specifically switch on a set of genes in venous cells. Both BMP4 and BMPR1a are enriched around veins, enabling this specificity. This is very helpful to know, as BMP inhibitors are already available and could be repurposed to target vessel growth. We have also now used this research to demonstrate the ETS factors are not sufficient for specific patterns of gene expression (e.g. vein-specific, artery-specific) within the endothelium. |
Exploitation Route | These results can be used in several ways: 1) to develop new treatments to prevent tumour vessel growth 2) to develop treatments to increase vessel growth during regeneration, 3) The animal models generated during this grant can now be used as tools to study the behaviour of this novel pathway in all cardiovascular pathologies. Some of this we are doing ourselves: the Ludwig institute is providing funding for research in cancer models, the BHF have provided funding to look at the behaviour of these pathways in the ischemic heart, and we have recieved pump prime funding to look at the behaviour of our enhancer:reporter animal models in cardiovascular diseases. We have also reported these findings at a number of international meetings, and have two papers were published with this information. Additionally, we are making the animal models generated by this grant available to anyone who is interested. |
Sectors | Healthcare |
Description | Academic Head Athena Swan |
Geographic Reach | Local/Municipal/Regional |
Policy Influence Type | Membership of a guideline committee |
Description | Athena |
Geographic Reach | Local/Municipal/Regional |
Policy Influence Type | Membership of a guideline committee |
Description | Ethical review |
Geographic Reach | Local/Municipal/Regional |
Policy Influence Type | Membership of a guideline committee |
Description | BHF Centre of Research Excellence, Oxford Pump Priming Grant |
Amount | £13,000 (GBP) |
Funding ID | RE/13/1/30181 |
Organisation | University of Oxford |
Sector | Academic/University |
Country | United Kingdom |
Start | 12/2015 |
End | 12/2016 |
Description | Delineating a regulatory pathway controlling coronary vessel formation |
Amount | £156,879 (GBP) |
Funding ID | PG/21/10704 |
Organisation | British Heart Foundation (BHF) |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 02/2022 |
End | 08/2023 |
Description | Determining the regulatory pathways controlling venous and lymphatic vessel growth and their role during heart development and regeneration |
Amount | £1,150,716 (GBP) |
Funding ID | FS/17/35/32929 |
Organisation | British Heart Foundation (BHF) |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 01/2018 |
End | 12/2023 |
Description | Fondation Leducq Transatlantic Network of Excellence |
Amount | $6,000,000 (USD) |
Organisation | The Leducq Foundation |
Sector | Charity/Non Profit |
Country | France |
Start | 01/2019 |
End | 01/2024 |
Description | University of Oxford John Fell Fund |
Amount | £49,395 (GBP) |
Organisation | University of Oxford |
Sector | Academic/University |
Country | United Kingdom |
Start | 08/2019 |
End | 01/2021 |
Title | Eph mouse |
Description | Transgenic mouse in which venous endothelial cells are marked by LacZ reporter gene |
Type Of Material | Model of mechanisms or symptoms - mammalian in vivo |
Year Produced | 2017 |
Provided To Others? | Yes |
Impact | none yet |
Title | coup mouse |
Description | Transgenic mice in which venous and lymphatic endothelial cells are labelled by LacZ reporter gene |
Type Of Material | Model of mechanisms or symptoms - mammalian in vivo |
Year Produced | 2017 |
Provided To Others? | Yes |
Impact | None yet |
Title | coup-fish |
Description | Zebrafish in which the venous endothelium is specifically expressing GFP |
Type Of Material | Model of mechanisms or symptoms - non-mammalian in vivo |
Year Produced | 2015 |
Provided To Others? | Yes |
Impact | This vein specific GFP fish provides an invaluable resource to many research groups |
Title | coup-plasmid |
Description | Enhancer that specifically drives gene expression in veins |
Type Of Material | Model of mechanisms or symptoms - non-mammalian in vivo |
Year Produced | 2013 |
Provided To Others? | Yes |
Impact | none yet |
Title | dll4-cre |
Description | Arterial and angiogenic specific Cre model |
Type Of Material | Model of mechanisms or symptoms - mammalian in vivo |
Year Produced | 2014 |
Provided To Others? | Yes |
Impact | None yet |
Title | ephb4 fish |
Description | fish expressing GFP in veins only |
Type Of Material | Model of mechanisms or symptoms - non-mammalian in vivo |
Year Produced | 2015 |
Provided To Others? | Yes |
Impact | Paper in preparation |
Description | George |
Organisation | University of Liverpool |
Department | Department of Musculoskeletal Biology |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We provide know-how in in-silico enhancer prediction, and generate Tol2-mediated zebrafish transgenics. |
Collaborator Contribution | George Bou-Gharios and his team at the Kennedy Institute (now part of University of Oxford) generate high-quality transgenic mice for us. |
Impact | We have two funded project grants utilizing data from this collaboration (MRC and BBSRC). We have three papers published as co-authors. |
Start Year | 2011 |
Description | Gillian |
Organisation | University of Oxford |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | I have collaborated with researchers within the Radcliffe Department of Medicine to expand our analysis of blood vessels into atherosclerotic models, and into models of vein grafting |
Collaborator Contribution | Provides the animal models required for this work |
Impact | Shared internal pilot grant |
Start Year | 2019 |
Description | Janice |
Organisation | University College London |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Providing enhancer:reporter mice with delineated regulatory pathways |
Collaborator Contribution | Conducting hind-limb ischemia |
Impact | none yet |
Start Year | 2019 |
Description | Nicola |
Organisation | University of Oxford |
Department | Department of Physiology, Anatomy and Genetics |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Providing specific transgenic mice Providing knowledge of transcriptional regulators of different endothelial cells within the heart |
Collaborator Contribution | Conducting cardiac injury, harvesting tissue |
Impact | BHF project grant awarded (PG/16/34/32135) Collaborator on Oxbridge BHF Centre of Regenerative Medicine application in 2017 The results formed part of the preliminary data in my BHF Senior Research Fellowship application Jan 2017 |
Start Year | 2015 |
Description | Rui |
Organisation | University of Oxford |
Department | Oxford Centre for Diabetes Endocrinology and Metabolism (OCDEM) |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Transgenic fish with different types of endothelial cells labeled by GFP |
Collaborator Contribution | Expertise and assistance in ATAC-seq |
Impact | Funded pump-prime grant from BHF Oxford CRE Provided ATAC-seq database for future enhancer identification |
Start Year | 2015 |
Description | Tim |
Organisation | University of Sheffield |
Department | School of Health and Related Research (ScHARR) |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Identified components of BMP pathway likely to be involved in venous identity in fish |
Collaborator Contribution | Used their recently developed endothelial-specific CRISPR-Cas9 fish to specifically delete specific components of BMP signaling within the vasculature |
Impact | Paper published in Nat Comms Collaborator in my recent BHF Senior Research Fellowship application |
Start Year | 2016 |
Description | Behind the headlines - Museum outreach |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | The Museum of Natural History in Oxford runs many activities to try and engage the public with scientific research, including their regular 'Super Science Saturdays' events. Last autumn, as part of a special themed Super Science Saturday called 'Behind The Headlines', a team of scientists from mine and Roger Patient's lab in the MRC Molecular Haematology Unit created a series of different activities to explain the science behind the headline: 'How tiny fish could hold the key to blood cancer treatment'. Over 2,500 visitors attended the event, which included numerous other activities. Approximately half of those in attendence were estimated to be school age |
Year(s) Of Engagement Activity | 2016 |
URL | https://wimmblog.com/2017/02/06/the-science-behind-the-headlines/ |
Description | Curiosity carnival |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Day long event "Curiosity Carnival" at Natural History Museum, part of a larger pan-European event to interest general public in science. Included stalls, games, posters, talks, movies etc. |
Year(s) Of Engagement Activity | 2017 |
Description | Public engagement in shopping centre |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | Day-long outreach activity at local shopping centre, including providing information and a game to engage general public. Seen by all at centre, engaged 1-1 with approximately 50 people. |
Year(s) Of Engagement Activity | 2017 |
Description | School visit Alice |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Presentation about cardiovascular system to year 6 childen, approximately 60 children |
Year(s) Of Engagement Activity | 2019 |
Description | Sun article |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Article about my research (and others) in The Sun, on the BBC science website and others, including image of genetically modified zebrafish. |
Year(s) Of Engagement Activity | 2022 |
Description | The Headington Festival |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Ran information stall at the Headington Festival, a local fate day attended by a large number of general public |
Year(s) Of Engagement Activity | 2019 |
Description | oxford open doors 2019 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Ran event as part of Oxford Open Doors, sparked many questions and discussions with wide range of people |
Year(s) Of Engagement Activity | 2019 |
Description | oxford science festival |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Participated in NDM presentation at festival, talked about research with public |
Year(s) Of Engagement Activity | 2015,2016 |
Description | work experience |
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 | Hosted 6th formers for work experience in our lab |
Year(s) Of Engagement Activity | 2015,2016 |