Regulation of VEGF splicing

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.

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.

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.