Regulation of VEGF splicing
Lead Research Organisation:
University of Nottingham
Department Name: Division of Pre-Clinical Oncology
Abstract
Vascular Endothelial Growth Factor is fundamental regulator of blood vessel growth (angiogenesis). It is responsible for blood vessel growth in all tissues in human development from the embryo to the newborn child. It is also key to growth of tissues as children develop into adults, and in adults it is necessary in normal physiology during tissue remodelling (e..g in muscle growth in response to exercise) and in many diseases including cancer, diabetes and heart disease . Over the last three years, our research group has discovered that the way that cells edit together the message encoded by the gene that codes for the most important growth factor for blood vessels (called VEGF) results in two different families of proteins called VEGFxxx and VEGFxxxb. The VEGFxxxb family is widely expressed in normal human tissues, but switched off during angiogenesis, when the VEGFxxx family take over. The VEGFxxx family stimulates blood vessel growth, whereas the VEGFxxxb stops vessels growing, and we have recently identified many new functions of this family - they protects cells from injury, stop development of pain, maintain kidney function, and control fertility. It is now clear that this alternative editing (splicing) is controlled by cells, and appears to switch during angiogenesis and other cell stresses. However, little is known about how cells balance expression of the two families of isoforms by regulating splicing of VEGF in normal tissues. This area is beyond the scope of the medical research charities currently supporting our work to find out whether these isoforms can be used to treat cancers, heart disease, diabetes, pain, renal failure and other conditions. This project aims to determine the molecular and cellular pathways that regulate the balance of splicing of the VEGF mRNA. It will identify some of the molecules that control these splicing events, determine the sequences within the VEGF gene that are recognised by splicing factors, and identify how and where this splicing balance alters in live animals. It will do so using a new, cutting edge technology of in vivo splice reporters. These are synthetic DNA sequences that code for green or red proteins depending on which forms are made. These sequences can be used to follow in real time in live cells which way splicing is happening. This is the only way that splicing can be followed within a live single cell in response to treatments, to normal function, or growth or changes in animals. These new sequences are unique to the laboratory in Bristol, and we wish to exploit this advantage over competing groups in the USA to generate advances in scientific understanding from a UK base.
Technical Summary
VEGF is a fundamental regulator of angiogenesis in all tissues in development and adulthood. VEGF comprises two families of isoforms generated by alternative splicing. VEGFxxx isoforms are pro-angiogenic. In 2002 we identified a sister family of isoforms that are anti-angiogenic, predominate in normal tissues but are down-regulated during angiogenesis when the pro-angiogenic isoforms take over, which has stimulated a program of research in our and other laboratories investigating their role in disease states and normal physiology. They have now been identified as cytoprotective agents for oxidative stress, hyperglycaemia, ischemia and hypoxia. They are anti-nociceptive, control glomerular permeability in the kidney, fertility, mammary development and regulate angiogenesis in physiological states. This alternative splicing is regulated at the cellular level, and switches during the angiogenesis process. We recently identified some of the mechanisms underlying regulation of alternate splicing of VEGF in vitro systems using simple cell treatments and assessment of splicing products. We have identified some sequences in the splice regions that specify splice site choice. This project will identify new molecular and cellular pathways that regulate this alternate splicing in vivo. It will make use of recently developed bichromatic splicing reporter systems to identify the molecular regulators and sequences recognised by these regulators. These reporters offer a unique advantage because they can be used for high throughput screening (e.g. genome wide siRNA screens), can follow splicing in real time in single cell populations (e.g. in circulating cells, or complex organs such as the glomerulus), and can be used to visualise splicing in intact animals. We will use these systems to determine the splicing switches that occur in in vivo physiological systems, by generating transgenic animals that have real time fluorescent bi-chromatic reporter splicing systems.
Planned Impact
This project will extend our understanding of one of the most basic physiological processes on which multi-cellular, multi-organ organisms depend. The process of developing and extending a vascular infrastructure that ensures appropriate delivery of nutrients to, and removal of toxic metabolites from, the tissues. This will be achieved by investigating the molecular mechanism that determines the VEGF mRNA splicing repertoire and therefore the time and spatially specific proteome that governs a physiological angiogenic phenotype. We will use molecular, cellular and animal models.
The methods utilised and optimised will be applicable to further study of angiogenic situations influenced by VEGF, both in in physiology, such as wound healing, and disease such as malignancy. Allowing detailed analysis of soluble mediators, factors, cytokines and splicing mediators and related drug targets.
Since over 90% of genes splice, with an average number of 7 isoforms, and since many physiological responses and human diseases depend on angiogenesis our findings may have a broad impact.
The methods utilised and optimised will be applicable to further study of angiogenic situations influenced by VEGF, both in in physiology, such as wound healing, and disease such as malignancy. Allowing detailed analysis of soluble mediators, factors, cytokines and splicing mediators and related drug targets.
Since over 90% of genes splice, with an average number of 7 isoforms, and since many physiological responses and human diseases depend on angiogenesis our findings may have a broad impact.
Organisations
People |
ORCID iD |
David Bates (Principal Investigator) |
Publications
Li L
(2022)
A repositioning screen using an FGFR2 splicing reporter reveals compounds that regulate epithelial-mesenchymal transitions and inhibit growth of prostate cancer xenografts.
in Molecular therapy. Methods & clinical development
Kitchen P
(2020)
A Runaway PRH/HHEX-Notch3-Positive Feedback Loop Drives Cholangiocarcinoma and Determines Response to CDK4/6 Inhibition.
in Cancer research
Mavrou A
(2014)
Abstract 2749: SRPK1 inhibition and modulation of VEGF alternative splicing as a potential therapeutic strategy in prostate cancer
in Cancer Research
Bowler E
(2019)
Alternative Splicing in Angiogenesis.
in International journal of molecular sciences
Stevens M
(2016)
Alternative Splicing in CKD.
in Journal of the American Society of Nephrology : JASN
Hamdollah Zadeh MA
(2015)
Alternative splicing of TIA-1 in human colon cancer regulates VEGF isoform expression, angiogenesis, tumour growth and bevacizumab resistance.
in Molecular oncology
Munkley J
(2019)
Androgen-regulated transcription of ESRP2 drives alternative splicing patterns in prostate cancer.
in eLife
Kikuchi R
(2019)
Anti-angiogenic isoform of vascular endothelial growth factor-A in cardiovascular and renal disease.
in Advances in clinical chemistry
Stevens M
(2018)
Assessment of Kidney Function in Mouse Models of Glomerular Disease.
in Journal of visualized experiments : JoVE
Description | We have discovered that we can develop pharmaceutical compounds that can reach the back of the eye as eye drops in large animal models that could be used for treatment of age related macular degeneration and diabetic retinopathy |
Exploitation Route | Development of eye drops for treatment of AMD> |
Sectors | Pharmaceuticals and Medical Biotechnology |
URL | http://exonate.com |
Description | The work done on this grant laid the foundation for the spin out of Exonate Ltd from the University of Nottingham. This is now a company that has raised>£6.5M in financing with 14 employees and drugs in development, and has signed a major collaboration agreement with Johnson and Johnson to fund further development of these eye drops in age related macular degeneration and diabetic retinopathy through clinical trial. |
First Year Of Impact | 2013 |
Sector | Pharmaceuticals and Medical Biotechnology |
Impact Types | Economic |
Description | BBSRC Pathfinder |
Amount | £11,708 (GBP) |
Funding ID | BB/P012035/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 10/2016 |
End | 03/2017 |
Description | Charity Funding |
Amount | £46,848 (GBP) |
Organisation | Richard Bright VEGF Research Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 05/2017 |
End | 04/2019 |
Description | Industry funded grant |
Amount | £232,239 (GBP) |
Organisation | Exonate |
Sector | Private |
Country | United Kingdom |
Start |
Description | MRC Confidence in Concept |
Amount | £70,000 (GBP) |
Organisation | Medical Research Council (MRC) |
Sector | Public |
Country | United Kingdom |
Start |
Description | Project grant |
Amount | £163,071 (GBP) |
Funding ID | 17/0005668 |
Organisation | Diabetes UK |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 07/2018 |
End | 06/2020 |
Title | Minigene |
Description | A minigene that can be used to assess VEGF splicing and the dependence of the splicing on teh sequence of the mRNA |
Type Of Material | Technology assay or reagent |
Year Produced | 2014 |
Provided To Others? | Yes |
Impact | Identification of the mechanism of binding of SRSF1 to VEGF |
Title | Splicing reporter mice |
Description | Transgenic mouse model harboring a VEGF splicing-sensitive fluorescent reporter |
Type Of Material | Model of mechanisms or symptoms - mammalian in vivo |
Year Produced | 2017 |
Provided To Others? | Yes |
Impact | This mouse model allows us to follow VEGF splicing in vivo in any cell in an organism. This is a great tool to assess whether compounds that affect VEGF splicing and may be therapeutic in diabetic nephropathy are changing splicing in normal tissues. |
Title | Splicing reporter tools |
Description | A plasmid that can tell us how mRNA is spliced between two isoforms of the VEGF gene. |
Type Of Material | Technology assay or reagent |
Year Produced | 2015 |
Provided To Others? | Yes |
Impact | Identification of novel anti-angiogenic agents |
Title | VEGF splicing reporter mouse |
Description | Transgenic mouse expressing VEGF fluorescent splicing reporter gen |
Type Of Material | Model of mechanisms or symptoms - mammalian in vivo |
Year Produced | 2016 |
Provided To Others? | No |
Impact | Mouse not published yet. |
Company Name | Exonate Ltd |
Description | Drug discovery company focussed on VEGF splicing control. Raised >£7M since start up. Won UK Business Angels Association Investment "One to watch" award 2017. |
Year Established | 2013 |
Impact | Closed seed round in March 2014. Employs 14 people in 2018 generating new drugs for age related macular degeneration and diabetic retinopathy. Nominated lead compound in 2018. In 2019 we closed a major funding deal from Janssen which will fund the company activities in eye disease including through clinical trial. The value of this license deal is potentially very large. In March 2020 we are moving laboratories out of the University of Nottingham to MediCity. |
Website | http://www.exonate.com |